Note: Descriptions are shown in the official language in which they were submitted.
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Novel therapeutic compositions for treating infection by Lawsonia spp.
FIELD OF THE INVENTION
The present invention relates generally to therapeutic compositions for the
treatment
and/or prophylaxis of intestinal disease conditions in animals and birds
caused or
exacerbated by Lawsonia intracellularis or similar or otherwise related
microorganism. In particular, the present invention provides a novel gene
derived
from L. intracellularis which encodes an immunogenic polypeptide. The
poiypeptide
described herein, selected from the group consisting of flhB, fliR, ntrC,
glnH, motA,
motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or
derivative of
any one or more of said polypeptides, is particularly useful as an antigen in
vaccine
preparation for conferring humoral immunity against L. intracellularis and
related
pathogens in animal hosts. The present invention is also directed to methods
for the
treatment and/or prophylaxis of such intestinal disease conditions and to
diagnostic
agents and procedures for detecting L. intracellularis or similar or otherwise
related
microorganisms.
GENERAL
Bibliographic details of the publications numerically referred to in this
specification are
collected at the end of the description. All patents, patent applications, and
publications cited herein are incorporated by reference in their entirety.
Reference hereinafter to "Lawsonia intracellularis" or its abbreviation "L.
intracellularis" includes all microorganisms similar to or otherwise related
to this
microorganism, as described by Stills (1991 ) or Jones et a1.(1997) or Lawson
et al.
(1993) or McOrist et al. (1995).
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References herein to "AGAL" shall be taken to mean a reference to the
Australian
Government Analytical Laboratories located at 1 Suakin Street, Pymble, New
South
Wales 2073, Australia. All biological deposits referred to herein in respect
of the
plasmids assigned AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH);
NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); and NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN) have been made under the provisions of
the Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure.
As used herein, the word "flh8", or the term "flh8 gene", shall be taken to
refer to a
gene encoding the antigenic-flhB polypeptide of the present invention, which
gene
comprises the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid pGTE#2 which
has
been deposited under AGAL Accession No. NM00/16477. The word '~IhB"or the
term "flh8 gene" shall further be taken to include a degenerate or
complementary
nucleotide sequence to SEQ ID NO: 1 or the nucleotide sequence of the L.
intracellularis gene contained in the plasmidpGTE#2 which has been deposited
under
AGAL Accession No. NM00/16477. It shall also be understood that the term "flhB
polypeptide" refers to a polypeptide of the invention which comprises the
amino acid
sequence set forth in SEQ ID NO: 2 or a polypeptide encoded by the L.
intracellularis
gene contained in the plasmid pGTE#2 which has been deposited under AGAL
Accession No. NM00/16477. The term "flhB polypeptide" shall further be taken
to
include a polypeptide which is functionally-related to or immunologically
cross-
reactive with the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by the
L.
intracellularis gene contained in the plasmid pGTE#2 which has been deposited
under AGAL Accession No. NM00/16477.
As used herein, the word "fliR", or the term "fliR gene", shall be taken to
refer to a
gene encoding the antigenic fliR polypeptide of the present invention, which
gene
comprises the nucleotide sequence set forth in SEQ ID NO: 3 or the nucleotide
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sequence of the L. intracellularis gene contained in the plasmid pGTE#3 which
has
been deposited under AGAL Accession No.NM00/16478. The word '~IiR"or the term
"fliR gene" shall further be taken to include a degenerate or complementary
nucleotide sequence to SEQ ID NO: 3, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#3 which has been deposited
under AGAL Accession No.NM00/16478. It shall also be understood that the term
"fliR polypeptide" refers to a polypeptide of the invention which comprises
the amino
acid sequence set forth in SEQ ID NO: 4 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#3 which has been deposited
under AGAL Accession No.NM00/16478. The term "fliR polypeptide" shall further
be
taken to include a polypeptide which is functionally-related to or
immunologically
cross-reactive with the polypeptide of SEQ ID NO: 4 or a polypeptide encoded
by the
L. intracellularis gene contained in the plasmid pGTE#3 which has been
deposited
under AGAL Accession No.NM00/16478.
As used herein, the word "ntrC", or the term "ntrC gene", shall be taken to
refer to a
gene encoding the antigenic ntrC polypeptide of the present invention, which
gene
comprises the nucleotide sequence set forth in SEQ ID NO: 5 or the nucleotide
sequence of the L, intracellularis gene contained in the plasmid pGTE#6 which
has
been deposited under AGAL Accession No.NM00/16481. The word "ntrC"or the term
"ntrC gene" shall further be taken to include a degenerate or complementary
nucleotide sequence to SEQ ID NO: 5, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#6 which has been deposited
under AGAL Accession No.NM00/16481. It shall also be understood that the term
"ntrC polypeptide" refers to a polypeptide of the invention which comprises
the amino
acid sequence set forth in SEQ ID NO: 6 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#6 which has been deposited
under AGAL Accession No.NM00/16481. The term "ntrC polypeptide" shall further
be taken to include a polypeptide which is functionally-related to or
immunologically
cross-reactive with the polypeptide of SEQ ID NO: 6 or a polypeptide encoded
by the
L. intracellularis gene contained in the plasmid pGTE#6 which has been
deposited
under AGAL Accession No.NM00/16481.
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MISSING AT THE TIME OF PUBLICATION
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under AGAL Accession No.NM00/16479 and which has homology to SEQ ID NO: 9.
The term "motA polypeptide" shall further be taken to include a polypeptide
which is
functionally-related to or immunologically cross-reactive with the polypeptide
of SEQ
ID NO: 10 or a polypeptide encoded by the L. intracellularis gene contained in
the
plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479
and having homology to SEQ ID NO: 9.
As used herein, the word "motB", or the term "mot8 gene", shall be taken to
refer to
a gene encoding the antigenic motB polypeptide of the present invention, which
gene
comprises the nucleotide sequence set forth in SEQ ID NO: 11 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid pGTE#4 which
has
been deposited under AGAL Accession No.NM00/16479 and having homology to
SEQ ID NO: 11. The word "mot8"or the term "mot8 gene" shall further betaken to
include a degenerate or complementary nucleotide sequence to SEQ ID NO: 11, or
the nucleotide sequence of the L. intracellularis gene contained in the
plasmid
pGTE#4 which has been deposited under AGAL Accession No.NM00/16479 and
having homology to SEQ ID NO: 11. It shall also be understood that the term
"motB
polypeptide" refers to a polypeptide of the invention which comprises the
amino acid
sequence set forth in SEQ ID NO: 12 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#4 which has been deposited
under AGAL Accession No.NM00/16479 and having homology to SEQ ID NO: 11.
The term "motB polypeptide" shall further be taken to include a polypeptide
which is
functionally-related to or immunologically cross-reactive with the polypeptide
of SEQ
ID NO: 12 or a polypeptide encoded by the L. intracellularis gene contained in
the
plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479
and having homology to SEQ ID NO: 11.
As used herein, the word "tlyC", or the term "tlyC gene", shall be taken to
refer to a
gene encoding the antigenic tlyC polypeptide of the present invention, which
gene
comprises the nucleotide sequence set forth in SEQ ID NO: 13 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid pGTE#5 which
has
been deposited under AGAL Accession No.NM00/16480. The word 'tlyC"or the term
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"tlyC gene" shall further be taken to include a degenerate or complementary
nucleotide sequence to SEQ ID NO: 13, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#5 which has been deposited
under AGAL Accession No.NM00/16480. It shall also be understood that the term
S "tlyC polypeptide" refers to a polypeptide of the invention which comprises
the amino
acid sequence set forth in SEQ ID NO: 14 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#5 which has been deposited
under AGAL Accession No.NM00/16480. The term "tlyC polypeptide" shall further
be
taken to include a polypeptide which is functionally-related to or
immunologically
cross-reactive with the polypeptide of SEQ ID NO: 14 or a polypeptide encoded
by
the L. intracellularis gene contained in the plasmid pGTE#5 which has been
deposited under AGAL Accession No.NM00/16480.
As used herein, the word "ytfM", or the term "ytfM gene", shall be taken to
refer to a
gene encoding the antigenic ytfM polypeptide of the present invention, which
gene
comprises the nucleotide sequence set forth in SEQ ID NO: 15 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid pGTE#7 which
has
been deposited under AGAL Accession No.NM00/16482. The word 'ytfM"or the term
"ytfM gene" shall further be taken to include a degenerate or complementary
nucleotide sequence to SEQ ID NO: 15, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#7 which has been deposited
under AGAL Accession No.NM00/16482. It shall also be understood that the term
"ytfM polypeptide" refers to a polypeptide of the invention which comprises
the amino
acid sequence set forth in SEQ ID NO: 16 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#7 which has been deposited
under AGAL Accession No.NM00/16482. The term "ytfM polypeptide" shall further
be taken to include a polypeptide which is functionally-related to or
immunologically
cross-reactive with the polypeptide of SEQ ID NO: 16 or a polypeptide encoded
by
the L. intracellularis gene contained in the plasmid pGTE#7 which has been
deposited under AGAL Accession No.NM00/16482.
As used herein, the word "ytfN", or the term "ytfN gene", shall be taken to
refer to a
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gene encoding the antigenic ytfN polypeptide of the present invention, which
gene
comprises the nucleotide sequence set forth in SEQ ID NO: 17 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid pGTE#8 which
has
been deposited under AGAL Accession No. NM01/23286. The word 'ytfN"or the
S term "ytfN gene" shall further be taken to include a degenerate or
complementary
nucleotide sequence to SEQ ID NO: 17 or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#8 which has been deposited
under AGAL Accession No. NM01/23286. It shall also be understood that the term
"ytfN polypeptide" refers to a polypeptide of the invention which comprises
the amino
acid sequence set forth in SEQ ID NO: 18 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#8 which has been deposited
under AGAL Accession No. NM01/23286. The term "ytfN polypeptide" shall further
be taken to include-a-polypeptide which is functionally-related to or
immunologically
cross-reactive with the polypeptide of SEQ ID NO: 18 or a polypeptide encoded
by
the L. intracellularis gene contained in the plasmid pGTE#8 which has been
deposited under AGAL Accession No. NM01/23286.
As used herein the words "from" or "of", and the term "derived from" shall be
taken
to indicate that a specified product, in particular a macromolecule such as a
polypeptide, protein, gene or nucleic acid molecule, antibody molecule, Ig
fraction,
or other macromolecule, or a biological sample comprising said macromolecule,
may
be obtained from a particular source, organism, tissue, organ or cell, albeit
not
necessarily directly from that source, organism, tissue, organ or cell.
Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising", will be
understood to
imply the inclusion of a stated step or element or integer or group of steps
or
elements or integers but not the exclusion of any other step or element or
integer or
group of elements or integers.
Those skilled in the art will appreciate that the invention described herein
is
susceptible to variations and modifications other than those specifically
described.
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It is to be understood that the invention includes all such variations and
modifications.
The invention also includes all of the steps, features, compositions and
compounds
referred to or indicated in this specification, individually or collectively,
and any and
all combinations or any two or more of said steps, features, compositions and
compounds.
The present invention is not to be limited in scope by the specific
embodiments
described herein, which are intended for the purposes of exemplification only.
Functionally equivalent products, compositions and methods are clearly within
the
scope of the invention, as described herein.
BACKGROUND OF THE INVENTION
The meat-producing sector of the agricultural industry is dependent upon tha
health
of its livestock and there is a need to maintain disease-free livestock for
human
consumption. The industry is subject to rapid economic downturn in response to
disease conditions adversely affecting livestock and the quality of meat
products
derived therefrom, including those diseases which may potentially be
transmitted to
humans. It is important, therefore, to have well defined treatments and
prophylactic
and diagnostic procedures available to deal with infections or potential
infections in
livestock animals and humans.
Meat products derived from porcine and avian species are significant
commercial
products in the agriculture industry. In particular, pigs form a major
component of the
meat industry. However, pigs are sensitive to a wide spectrum of intestinal
diseases
collectively referred to as porcine proliferative enteropathy (PPE). These
diseases
have previously been known as intestinal adenomatosis complex (Barker and van
Drumel, 1985), porcine intestinal adenomatosis (PIA), necrotic enteritis
(Rowland and
Lawson, 1976), proliferative haemorrhagic enteropathy (Love and Love, 1977),
regional ileitis (Jonsson and Martinsson, 1976), haemorrhagic bowel syndrome
(O'Neil, 1970), porcine proliferative enteritis and Campylobacter spp -
induced
enteritis (Straw, 1990).
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There are two main forms of PPE: a non-haemorrhagic form represented by
intestinal
adenomatosis which frequently causes growth retardation and mild diarrhoea;
and
a haemorrhagic form, which is often fatal, represented by proliferative
haemorrhagic
enteropathy (PHE), where the distal small intestine lumen becomes engorged
with
blood. PPE has been reported in a number of animal species including pigs
(McOrist
et al, 1993), hamsters (Stills, 1991 ), ferrets (Fox et al, 1989), guinea pigs
(Elwell et
al, 1981 ), rabbits (Schodeb and Fox, 1990) as well as avian species (Mason et
al,
1998).
PPE is a significant cost component associated with the pig industry,
especially in
terms of stock losses, medication costs, reduced growth rates of pigs and
increased
feed costs. PPE also contributes to downstream indirect costs in, for example,
additional labour costs and environmental costs in dealing with-antibiotic-
residue
contamination, and in control measures to prevent the organism from being
passed
on or carried to other animals or humans.
L, intracellularis is a causative agent of PPE (McOrist et al, 1995). L.
intracellularis
is an intracellular, possibly obligate intracellular, bacterium. It can only
be cultured
in vitro with tissue culture cells (Jones et al., 1997; Lawson et al., 1993;
McOrist et
al, 1995; International Patent Application No. PCT/US96/09576). L.
intracellularis is
located in the cytoplasm of the villus cells and intestinal crypt cells of
infected
animals. Pigs suffering from PPE are characterised by irregularities in the
villus cells
and intestinal crypt structure with epithelial cell dysplasia, wherein crypt
abscesses
form as the villi and intestinal crypts become branched and fill with
inflammatory cells.
Current control strategies for PPE rely on the use of antibacterials. However,
such
a strategy is considered to only be short to medium term, especially since
governmental regulatory pressures tend to discourage animal husbandry
practices
which involve the use of prophylactic antibiotics. There is a need, therefore,
to
develop effective, safe and low cost alternatives to the use of antibiotics
and, in
particular, to develop vaccine preparations capable of conferring protective
immunity
against L. intracellularis infection in livestock animals.
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The most effective vaccine preparations are generally comprised of a highly
antigenic
component, such as a polypeptide or other macromolecule which is derived from
the
pathogenic organism against which the vaccine is directed, wherein said
antigenic
component produces little or no contraindications when administered to a
susceptible
host animal, and produces little or no antigenic cross-reactivity with
desirable
organisms, such as non-pathogenic organisms that are a part of the normal
flora of
the intestinal tract or other tissues of said host animal. In summary, an
effective
vaccine preparation must be immunogenic, specific and safe.
Accordingly, there is a need to identify highly immunogenic antigens produced
by the
bacterium L. intracellularis.
International Patent Application No. PCT/AU96/00767 describes several L.
intracellularis partial genetic sequences, and partial polypeptides encoded
thereby.
However, there is a need to further identify polypeptide immunogens produced
by
the bacterium L. intracellularis and immunogenic peptides derived therefrom,
including those immunogens which are genus- or species-specific, for use in
improved vaccine compositions. The presently-described invention provides such
immunogens.
SUMMARY OF THE INVENTION
One aspect of the present invention is directed to an isolated or recombinant
immunogenic polypeptide which comprises, mimics or cross-reacts with a B-cell
or
T-cell epitope of a polypeptide derived from Lawsonia spp, in particular a
polypeptide
selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM,
and ytfN polypeptides, or a homologue, analogue or derivative of any one or
more
of said polypeptides.
Preferably, the isolated or recombinant immunogenic polypeptide is selected
from the
group consisting of the following:
(i) a polypeptide which comprises an amino acid sequence which has at
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least about 60% sequence identity overall to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(ii) a polypeptide which comprises an amino acid sequence which has at
least about 60% sequence identity overall to an amino acid sequence
encoded by L. infracellularis DNA contained within a plasmid selected from the
group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a polypeptide which comprises at least about 5 contiguous amino acids
of an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(iv) a polypeptide which comprises at least about 5 contiguous amino acids
of an amino acid sequence encoded by L. intracellularis DNA contained within
a plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B);
NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC);
NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8
ytfN); and
(v) a homologue, analogue or derivative of any one of (i) to (iv) which
mimics a B-cell or T-cell epitope of Lawsonia spp.
In an alternative preferred embodiment, the isolated or recombinant
immunogenic
polypeptide is selected from the group consisting of the following:
(i) a polypeptide which comprises an amino acid sequence encoded by a
nucleotide sequence having at least about 60% sequence identity overall to
a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, and 17;
(ii) a polypeptide which comprises an amino acid sequence encoded by a
nucleotide sequence having at least about 60% sequence identity overall to
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the nucleotide sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480
(plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482
(plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a polypeptide encoded by at least about 15 contiguous nucleotides of
a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, and 17;
(iv) a polypeptide encoded by at least about 15 contiguous nucleotides of
a nucleotide sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480
(plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482
(plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and
(v) a homologue, analogue or derivative of any one of (i) to (iv) which
mimics a B-cell or T-cell epitope of Lawsonia spp.
In a particularly preferred embodiment, the polypeptide of the present
invention
comprises or consists of an amino acid sequence selected from the group
consisting
of:
(i) an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; and
(ii) an amino acid sequence encoded by L. intracellularis DNA contained
within a deposited plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid
pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid
pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481
(plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and
NM01/23286 (plasmid pGTE#8 ytfN).
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A further aspect of the present invention provides a vaccine composition for
the
prophylaxis or treatment of infection in an animal, such as a pig or bird, by
L.
intracellularis or a similar or otherwise related microorganism, said vaccine
composition comprising an immunologically effective amount of an immunogenic
component which comprises an isolated or recombinant polypeptide selected from
the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN
polypeptides as described herein and one or more carriers, diluents and/or
adjuvants
suitable for veterinary or pharmaceutical use.
A further aspect of the invention extends to an immunologically interactive
molecule,
such as an antibody or antibody fragment, which is capable of binding to an
immunogenic polypeptide of the invention selected from the group consisting of
flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.
A further aspect of the invention provides a method of diagnosing infection of
an
animal by L. intracellularis or a related microorganism, said method
comprising the
steps of contacting a biological sample derived from said animal with an
immunologically interactive molecule of the present invention for a time and
under
conditions sufficient for a complex, such as an antigen:antibody complex, to
form,
and then detecting said complex formation.
A further aspect of the present invention contemplates a method of determining
whether or not an animal has suffered from a past infection, or is currently
infected,
by L. intracellularis or a related microorganism, said method comprising
contacting
a tissue or fluid sample, such as blood or serum derived from said animal,
with an
immunogenic polypeptide selected from the group consisting of flhB, fliR,
ntrC, glnH,
motA, motB, tlyC, ytfM, and ytfN polypeptides, or a peptide derived therefrom,
for a
time and under conditions sufficient for a complex, such as an
antigen:antibody
complex, to form, and then detecting said complex formation.
A further aspect of the present invention provides an isolated nucleic acid
molecule
which comprises a sequence of nucleotides that encodes, or is complementary to
a
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nucleic acid molecule that encodes, a polypeptide selected from the group
consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides,
including any
and all genes selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN genes as defined hereinabove.
In a preferred embodiment, the isolated nucleic acid molecule comprises a
nucleotide
sequence encoding a polypeptide that is immunologically cross-reactive with L.
intracellularis or other causative agent of PPE, wherein said nucleotide
sequence is
selected from the group consisting of:
(i) a nucleotide sequence having at least about 60% sequence identity
overall to a nucleotide sequence selected from the group consisting of SEQ
ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
(ii) a nucleotide sequence-having at least about 60% sequence identity
overall to L. intracellularis DNA contained within a plasmid selected from the
group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a nucleotide sequence which comprises at least about 15 contiguous
nucleotides of a sequence selected from the group consisting of SEQ ID NOs:
1, 3, 5, 7, 9, 11, 13, 15, and 17;
(iv) a nucleotide sequence which comprises at least about 15 contiguous
nucleotides of L. intracellularis DNA contained within a plasmid selected from
the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(v) a nucleotide sequence which hybridizes under at least low stringency,
more preferably moderate stringency, and most preferably high stringency
conditions to a nucleotide sequence selected from the group consisting of
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SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 or a complementary nucleotide
sequence thereto;
(vi) a nucleotide sequence which hybridizes under at least low stringency,
more preferably moderate stringency, and most preferably high stringency
conditions to L. intracellularis DNA contained within a plasmid selected from
the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and
(vii) a homologue, analogue or derivative of any one of (i) to (vi) which
encodes a polypeptide which mimics a B-cell or T-cell epitope of Lawsonia
spp.
In a particularly preferred embodiment, the isolated nucleic acid molecule of
the
present invention comprises or consists of a nucleotide sequence selected from
the
group consisting of:
(i) a nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1,3, 5, 7, 9, 11, 13, 15, and 17; or a degenerate variant thereof;
(ii) a nucleotide sequence of the L, intracellularis DNA contained within a
deposited plasmid selected from the group consisting of AGAL Accession
o Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2
flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN); and
(iii) a nucleotide sequence that encodes the same polypeptide as (i) or (ii),
wherein said polypeptide is selected from the group consisting of flhB, fliR,
ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN;
(iv) a nucleotide sequence that is complementary to (i) or (ii) or (iii); and
(v) a nucleotide sequence that hybridises under high stringency conditions
to the complement of a sequence selected from the group consisting of: SEQ
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ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and 17, wherein said nucleotide sequence is
the complement of a sequence that encodes a polypeptide that is
immunologically cross-reactive to a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN.
A still further aspect of the invention provides a diagnostic method of
detecting L.
intracellularis or related microorganism in a biological sample derived from
an animal
subject, said method comprising the steps of hybridising one or more
polynucleotide
or oligonucleotide probes or primers derived from a gene selected from the
group
consisting of flh8, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes,
or a
homologue, analogue or derivative thereof, to said sample, and then detecting
said
hybridisation using a detection means. The detection means according to this
aspect
of the invention is any nucleic acid-based hybridisation or amplification
reaction.
A further aspect of the invention provides an isolated probe or primer derived
from
a gene selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC,
ytfM, and ytfN genes. In a particularly preferred embodiment, the probe or
primer of
the invention is useful for isolating the ytfM and/or ytfN genes described
herein. More
preferably, the probe or primer of the invention comprises a nucleotide
sequence
selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 68 or a
complementary nucleotide sequence thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a copy of a photographic representation showing expression of
recombinant YtfN protein. The 5' portion of the gene up to the Bglll site was
cloned
into pET-30a. A plasmid with the fragment inserted in the proper orientation
was
transformed into E. coli BL21 (DE3) cells, and a single clone was propagated.
Induction was at OD62s=2.9 with 0.1 mM IPTG. Lane 1, whole cell lysate (WCL)
from
uninduced cells; lanes 2 and 3, WCL at 2.25 and 3 hrs post-induction,
respectively.
Arrow indicates the position of recombinant YtfN protein.
DETAILED DESCRIPTION OF THE INVENTION
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In work leading up to the present invention, the inventors sought to identify
immunogenic proteins of L. intracellularis for use in vaccines for the
prophylaxis and
treatment of PPE in animals, including pigs and birds.
Accordingly, one aspect of the present invention is directed to an isolated or
recombinant immunogenic polypeptide which comprises, mimics or cross-reacts
with
a B-cell or T-cell epitope of a polypeptide derived from Lawsonia spp,
selected from
the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN, or a
homologue, analogue or derivative of any one or more of said polypeptides.
Epitopes of Lawsonia spp. may be B cell epitopes or T-cell epitopes. It is
well-known
that antibody-binding sites (B-cell epitopes) involve linear as well as
conformational
epitopes (van Regenmortel, 1992). B-cell epitopes are predominantly-
conformational.
In contrast, T-cells recognize predominantly linear epitope sequences in
combination
with MHC class II molecules.
A precise identification and careful selection of epitopes of Lawsonia spp.
facilitates
the development of diagnostic reagents and vaccine compositions for the
effective
treatment or prophylaxis of Lawsonia infections. Epitope identification and
characterization (i.e., determination of the molecular weight, amino acid
sequence,
and structure of epitopes of Lawsonia spp.) may be performed using art-
recognised
techniques. For the detection of conformational epitopes, degrading and
denaturing
of the epitope molecule must be avoided in order to conserve the three-
dimensional
structure, because the antigen-antibody reaction will be diminished if the
secondary
structure of the epitope is altered significantly. In practice, the
characterisation and
isolation of linear non-conformational epitopes is easier, because any
immunoreactive regions are contained within a single polypeptide or peptide
fragment
which is capable of being purified under a range of conditions.
Both non-conformational and conformational epitopes may be identified by
virtue of
their ability to bind detectable amounts of antibodies (such as IgM or IgG)
from sera
of animals immunised against or infected with Lawsonia spp. and, in particular
L.
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infracellularis, or an isolated polypeptide derived therefrom or,
alternatively, by virtue
of their ability to bind detectable amounts of antibodies in a purified Ig
fraction derived
from such sera. The antibodies may be derived from or contained within pools
of
polyclonal sera, or may be monoclonal antibodies. Antibody fragments or
recombinant antibodies, such as those expressed on the surface of a
bacteriophage
or virus particle, such as in a phage display library, may also be employed.
The determination of T-cell epitopes is performed by analysing the ability of
the
epitope peptides to induce the proliferation of peripheral blood lymphocytes
or T-cell
clones. The identification of T-cell epitopes is accomplished using a variety
of
methods as known in the art, including the use of whole and fragmented native
or
recombinant antigenic protein, as well as the more commonly employed
"overlapping
peptide" method. in the latter method, overlapping peptides which span the
entire
sequence of a poiypeptide derived from Lawsonia spp. are synthesized and
tested
for their capacity to stimulate T-cell cytotoxic or proliferative responses in
vitro.
Structure determination of both conformational non-linear and non-
conformational
linear epitopes may be performed by nuclear magnetic resonance spectroscopy
(NMR) and X-ray crystallographic analysis. The determination of epitopes using
X-ray
techniques requires the protein-antibody complex to be crystallized, whereas
NMR
allows analysis of the complex in a liquid state. NMR measures the amount of
amino
acids as well as the neighbourhood of protons of different amino acid
residues,
wherein the alternating effect of two protons along the carbon backbone is
characteristic of a particular epitope.
A successful method to recognize non-conformational linear epitopes is the
immunoblot and in particular, the Western blot. Peptides may be generated from
a
complete Lawsonia spp. polypeptide by digestion with site-specific proteases,
such
as trypsin or chymotrypsin, and the peptides generated thereby can be
separated
using standard electrophoretic or chromatographic procedures. For example,
after
electrophoresis according to molecular weight using SDS/PAGE
(SDS/polyacrylamide gel electrophoresis) and/or according to isoelectric point
using
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IEF (isoelectric focussing) or alternatively, by two-dimensional
electrophoresis, the
peptides can be transferred to immobilizing nylon or nitrocellulose membranes
and
incubated with sera raised against the intact polypeptides. Peptides that
comprise
immunogenic regions (i.e., B-cell or T-cell epitopes) are bound by the
antibodies in
the sera and the bound antibodies may be detected using secondary antibodies,
such as anti-IgG antibodies, that have been labelled radioactively or
enzymatically.
The epitopes may then be characterised by purification based upon their size,
charge
or ability to bind specifically to antibodies against the intact polypeptide,
using one
or more techniques, such as size-exclusion chromatography, ion-exchange
chromatography, affinity chromatography or ELISA among others. After
purification
of the epitope, only one band or spot should be detectable with gel
electrophoresis.
The N-terminal or total sequencing of the polypeptide or peptide fragment
offers the
possibility to compare the amino acid sequence with known proteins in
databases.
Several computer-driven algorithms have now been devised to search for T-cell.
epitopes in proteins (Margalit et al, 1987; Vajda and C. DeLisi, 1990; Altuvia
et al.,
1994; Parker et a1.1994; DeGroot ef a1.,1995; Gabriel et al, 1995; Meister et
al.,
1995). These algorithms search the amino acid sequence of a given protein for
characteristics believed to be common to immunogenic peptides, locating
regions
that are likely to induce a cellular immune response in vitro. Computer-driven
algorithms can identify regions of a Lawsonia spp. polypeptide that contain
epitopes
and are less variable among different isolates. Alternatively, computer-driven
algorithms can rapidly identify regions of each isolates more variable
proteins that
should be included in a multivalent vaccine.
The AMPHI algorithm (Margalit et a1.,1987), which is based on the periodicity
of T cell
epitopes, has been widely used for the prediction of T-cell antigenic sites
from
sequence information alone. Essentially, AMPHI describes a common structural
pattern of MHC binding motifs, since MHC binding motifs (i.e., patterns of
amino
acids that appear to be common to most of the peptides that bind to a specific
MHC
molecule) appear to exhibit the same periodicity as an alpha helix.
Identification of
T-cell epitopes by locating MHC binding motifs in an amino acid sequence
provides
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an effective means of identifying immunogenic epitopes in diagnostic assays.
The EpiMer algorithm (Meister et al., 1995; Gabriel et al., 1995; DeGroot et
al., 1995)
locates clustered MHC binding motifs in amino acid sequences of proteins,
based
upon the correlation between MHC binding motif-dense regions and peptides that
may have the capacity to bind to a variety of MHC molecules (promiscuous or
multi-determinant binders) and to stimulate an immune response in these
various
MHC contexts as well (promiscuous or multi-determinant epitopes). The EpiMer
algorithm uses a library of MHC binding motifs for multiple class I and class
II HLA
alleles to predict antigenic sites within a protein that have the potential to
induce an
immune response in subjects with a variety of genetic backgrounds. EpiMer
locates
matches to each MHC-binding motif within the primary sequence of a given
protein
antigen. The relative density of these motif matches is determined along the
iength-
of the antigen, resulting in the generation of a motif-density histogram.
Finally; the
algorithm identifies protein regions in this histogram with a motif match
density above
an algorithm-defined cutoff density value, and produces a list of subsequences
representing these clustered, or motif-rich regions. The regions selected by
EpiMer
may be more likely to act as multi-determinant binding peptides than randomly
chosen peptides from the same antigen, due to their concentration of MHC-
binding
motif matches. The selection of regions that are MHC binding motif-dense
increases
the likelihood that the predicted polypeptide or peptide fragment contains a
"valid"
motif, and furthermore, that the reiteration of identical motifs may
contribute to
binding.
Additional MHC binding motif-based algorithms have been described by Parker et
a1.(1994) and Altuvia et a1.(1994). In these algorithms, binding to a given
MHC
molecule is predicted by a linear function of the residues at each position,
based on
empirically defined parameters, and in the case of the Altuvia et a1.(1994)
algorithm,
known crystallographic structures may also be taken into consideration.
Recombinant methods offer the opportunity to obtain well characterized
epitopes of
high purity for the production of diagnostic reagents and epitope-specific
vaccine
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formulations (Mohapatra et al., 1995). Based upon the amino acid sequence of a
linear epitope and identification of the corresponding nucleotide sequence
encoding
same, polymerase chain reaction (PCR) may be performed to amplify the epitope-
encoding region from cDNA. After cloning and expression in a suitable
vector/host
system, a large amount of epitopes of high purity can be extracted.
Accordingly, the
present invention clearly extends to both isolated non-recombinant
polypeptides and
recombinant polypeptides in an impure or isolated form.
The term "polypeptide" as used herein shall be taken to refer to any polymer
consisting of amino acids linked by covalent bonds and includes within its
scope the
full-length amino acids disclosed herein, and any parts or fragments thereof
such as,
for example, peptides consisting of about 5-50 amino acid residues in length,
preferably about 5-30 amino acid residues in length, more preferably about 5-
20
amino acid residues in length, and even more preferably about 5-10 amino acid
residues in length. Also included within the scope of the definition of a
"polypeptide"
are amino acid sequence variants, containing one or more preferably
conservative
amino acid substitutions, deletions, or insertions, which do not alter at
least one
essential property of said polypeptide such as, for example, its
immunogenicity, use
as a diagnostic reagent, or effectiveness as a vaccine against Lawsonia spp,
amongst others. Accordingly, a polypeptide may be isolated from a source in
nature,
or chemically synthesized. Furthermore, a polypeptide may be derived from a
full-
length protein by chemical or enzymatic cleavage, using reagents such as CNBr,
trypsin, or chymotrypsin, amongst others.
Conservative amino acid substitutions are well-known in the art. For example,
one
or more amino acid residues of a native flagellar hook protein of the present
invention
can be substituted conservatively with an amino acid residue of similar
charge, size
or polarity, with the resulting polypeptide retaining an ability to function
in a vaccine
or as a diagnostic reagent as described herein. Rules for making such
substitutions
include those described by Dayhof (1978). More specifically, conservative
amino acid
substitutions are those that generally take place within a family of amino
acids that
are related in their side chains. Genetically-encoded amino acids are
generally
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divided into four groups: (1 ) acidic=aspartate, glutamate; (2) basic=lysine,
arginine,
and histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, and tryptophan; and (4) uncharged polar= glycine,
asparagine, glutamine, cysteine, serine, threonine, and tyrosine.
Phenylalanine,
tyrosine and tryptophan are also jointly classified as aromatic amino acids.
One or
more replacements within any particular group such as, for example, the
substitution
of leucine for isoleucine or valine or alternatively, the substitution of
aspartate for
glutamate or threonine for serine, or of any other amino acid residue with a
structurally-related amino acid residue, will generally have an insignificant
effect on
the function of the resulting polypeptide.
The present invention is not limited by the source of the subject immunogen
and
clearly extends to isolated and recombinant polypeptides which are derived
from a
natural or a non-natural occurring source.
The term "recombinant polypeptide" as used herein shall be taken to refer to a
polypeptide which is produced in vitro or in a host cell by the expression of
a genetic
sequence encoding said polypeptide, which genetic sequence is under the
control of
a suitable promoter, wherein a genetic manipulation has been performed in
order to
achieve said expression. Accordingly, the term "recombinant polypeptide"
clearly
encompasses polypeptides produced by the expression of genetic sequences
contained in viral vectors, cosmids or plasmids that have been introduced into
prokaryotic or eukaryotic cells, tissues or organs. Genetic manipulations
which may
be used in this context will be known to those skilled in the art and include,
but are
not limited to, nucleic acid isolation, restriction endonuclease digestion,
exonuclease
digestion, end-filling using the Klenow fragment of E. coli DNA polymerise I
or T4
DNA polymerise enzymes, blunt-ending of DNA molecules using T4 DNA
polymerise or Exolll enzymes, site-directed mutagenesis, ligation, and
amplification
reactions. As will be known to those skilled in the art, additional techniques
such as
nucleic acid hybridisations and nucleotide sequence analysis may also be
utilised in
the preparation of recombinant polypeptides, in confirming the identity of a
nucleic
acid molecule encoding a desired recombinant polypeptide and a genetic
construct
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comprising the nucleic acid molecule.
Wherein the polypeptide of the present invention is a recombinant polypeptide,
it may
be produced in and, if desirable, isolated from a recombinant viral vector
expression
system or host cell. As will be known to those skilled in the relevant art, a
cell for
production of a recombinant polypeptide is selected on the basis of several
parameters including the genetic constructs used to express the polypeptide
under
consideration, as well as the stability and activity of said polypeptide. It
will also be
known to those skilled in the art that the stability or activity of a
recombinant
polypeptide may be determined, at least in part, by post-translational
modifications
to the polypeptide such as, for example, glycosylation, acylation or
alkylation
reactions, amongst others, which may vary between cell lines used to produce
the
recombinant polypeptide.
Accordingly, in a more particularly preferred embodiment, the present
invention
extends to a recombinant polypeptide or a derivative, homologue or analogue
thereof
as present in a virus particle, or as produced in prokaryotic or eukaryotic
host cell, or
in a virus or cell culture thereof.
The present invention also extends to a recombinant polypeptide according to
any
of the foregoing embodiments which is produced in a bacterial cell belonging
to the
genus Lawsonia, in particular a cell of L. intracellularis, or a culture
thereof.
The term "isolated polypeptide" refers to a polypeptide of the present
invention which
has been purified to some extent, preferably to at least about 20% by weight
of
protein, preferably to at least about 50% by weight of protein, more
preferably to at
least about 60% by weight of protein, still more preferably to at least about
70% by
weight of protein and even more preferably to at least about 80% by weight of
protein
or greater, from its natural source or, in the case of non-naturally-occurring
polypeptides, from the culture medium or cellular environment in which it was
produced. Such isolation may be performed to improve the immunogenicity of the
polypeptide of the present invention, or to improve the specificity of the
immune
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response against that polypeptide, or to remove toxic or undesirable
contaminants
therefrom. The necessary or required degree of purity of an isolated
polypeptide will
vary depending upon the purpose for which the polypeptide is intended, and for
many
applications it will be sufficient for the polypeptide preparation to contain
no
contaminants which would reduce the immunogenicity of the polypeptide when
administered to a host animal, in particular a porcine or avian animal being
immunized against PPE or, alternatively, which would inhibit immuno-specific
binding
in an immunoassay for the diagnosis of PPE or a causative agent thereof.
The purity of an isolated polypeptide of the present invention may be
determined by
any means known to those skilled in the art, including the degree of
homogeneity of
a protein preparation as assessed by SDSlpolyacrylamide gel electrophoresis, 2-
dimensional electrophoresis, or amino acid composition analysis-or sequence
analysis.
Preferably, the polypeptide of the present invention will be substantially
homogeneous or substantially free of nonspecific proteins, as assessed by
SDS/polyacrylamide gel electrophoresis, 2-dimensional electrophoresis, or
amino
acid composition analysis or sequence analysis.
The polypeptide of the present invention can be purified for use as a
component of
a vaccine composition by any one or a combination of methods known to those of
ordinary skill in the art, including, for example, reverse phase
chromatography, HPLC,
ion-exchange chromatography, and affinity chromatography, among others.
In a preferred embodiment, the isolated or recombinant polypeptide of the
invention
functions is secretable into the periplasmic space of a cell, preferably into
the
periplasm of a prokaryotic cell, such as, for example, Escherichia coli. or L.
intracellularis, or, alternatively, is immunologically cross-reactive with a
L.
intracellularis polypeptide selected from the group consisting of flhB, fliR,
ntrC, glnH,
motA, motB, tlyC, ytfM, and ytfN.
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In a particularly preferred embodiment, the isolated or recombinant
polypeptide of the
invention is derived from Lawsonia spp. or other pathogenic agent associated
with
the onset and/or development of PPE and more preferably, the subject
polypeptide
is derived from L. intracellularis.
A B-cell or T-cell epitope of a polypeptide selected from the group consisting
of flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a
homologue,
analogue or derivative of any one or more of said polypeptides, may comprise
one
or more of the following:
(i) the primary. amino acid sequence of any one of said polypeptides, as
determined by an art-accepted methodology to comprise a continuous
non-conformational epitope;
(ii) the secondary structure which any one of said polypeptides adopt, as
determined by an art-accepted methodology to comprise a continuous
conformational epitope;
(iii) the tertiary structure which any one of said polypeptides adopt in
contact with another region of the same polypeptide molecule, as
determined by an art-accepted methodology to comprise a
discontinuous conformational epitope; or
(iv) the quaternary structure which any one of said polypeptides adopt in
contact with a region of another polypeptide molecule, as determined
by an art-accepted methodology to comprise a discontinuous
conformational epitope.
Accordingly, immunogenic polypeptides or derivatives, homologues or analogues
thereof comprising the same, or substantially the same primary amino acid
sequence
are hereinafter defined as "immunogens which comprise a B-cell or T-cell
epitope"
or similar term.
Immunogenic polypeptides or derivatives, homologues, or analogues thereof
comprising different primary amino acid sequences may comprise immunologically
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identical immunogens, because they possess conformational B-cell or T-cell
epitopes
that are recognised by the immune system of a host species to be identical.
Such
immunogenic polypeptides or derivatives, homologues or analogues thereof are
hereinafter defined as "immunogens which mimic or cross-react with a B-cell or
T-cell
epitope", or similar term.
Accordingly, the present invention extends to an immunogen which comprises,
mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or
recombinant
polypeptide according to any one of the foregoing embodiments or a derivative,
homologue or analogue thereof. In a particularly preferred embodiment, the
present
invention provides an immunogen which comprises, mimics, or cross-reacts with
a
B-cell or T-cell epitope of an isolated or recombinant polypeptide which in
its native
form is obtainable from a species of Lawsonia such as, but not limited to L.
intracellularis and which polypeptide preferably has the same biological
function as
a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN, as hereinbefore defined.
Preferably, such immunogenic polypeptides will not comprise a primary amino
acid
sequence which is highly-conserved between L. intracellularis and another non-
pathogenic microorganism which is normally resident in the gut or other organ
of an
animal, in particular a porcine or avian animal. The significance of this
exclusion to
those embodiments of the invention wherein specificity is essential to
performance
(eg vaccine and diagnostic applications) will be apparent to those skilled in
the art.
To improve the immunogenicity of a subject polypeptide of the present
invention one
or more amino acids not corresponding to the original protein sequence can be
added to the amino or carboxyl terminus of the polypeptide. Such extra amino
acids
are useful for coupling the polypeptide to another peptide or polypeptide, to
a large
carrier protein or to a solid support. Amino acids that are useful for these
purposes
include but are not limited to tyrosine, lysine, glutamic acid, aspartic acid,
cysteine
and derivatives thereof. Additional protein modification techniques can be
used such
as, e.g., NH2-acetylation or COOH-terminal amidation, to provide additional
means
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for coupling the polypeptide to another polypeptide or peptide molecule, or to
a solid
support. Procedures for coupling polypeptides to each other, or to carrier
proteins
or solid supports, are well known in the art. Polypeptides containing the
above-
mentioned extra amino acid residues at either the carboxyl- or amino-termini
and
either uncoupled or coupled to a carrier or solid support, are consequently
within the
scope of the present invention.
Furthermore, the polypeptide can be immobilised to a polymeric carrier or
support
material.
In an alternative embodiment, the immunogenicity of a polypeptide of the
present
invention may be improved using molecular biology techniques to produce a
fusion
protein containing one or more polypeptides of the present invention fused to
a
carrier molecules such as a highly immunogenic protein. For example, a fusion
protein containing a polypeptide of the present invention fused to the highly
immunogenic B subunit of cholera toxin can be used to increase the immune
response to the polypeptide. The present invention also contemplates fusion
proteins comprising a cytokine, such as an interleukin, fused to the subject
polypeptide of the present invention, and genes encoding same.
Preferably, the polypeptide of the present invention, or a derivative,
homologue or
analogue thereof, when administered to a mammal, induces an immune response
in said mammal. More preferably, the polypeptide of the present invention,
when
administered to a mammal, in particular a porcine animal (e.g., a pig) induces
a
protective immune response against Lawsonia spp., and preferably against L.
intracellularis, therein. As used herein, the phrase "induction of a
protective immune
response", and the like, refers to the ability of the administered polypeptide
of the
present invention to prevent or detectably slow the onset, development, or
progression of symptoms associated with Lawsonia infection, and preferably, to
prevent or detectably slow the onset, development; or progression of symptoms
associated with PPE in pigs.
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Preferably, the isolated or recombinant immunogenic polypeptide is selected
from the
group consisting of the following:
(i) a polypeptide which comprises an amino acid sequence which has at
least about 60% sequence identity overall to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(ii) a polypeptide which comprises an amino acid sequence which has at
least about 60% sequence identity overall to an amino acid sequence
encoded by L. intracellularis DNA contained within a plasmid selected from the
group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a polypeptide which comprises at least about 5 contiguous amino acids,
of an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(iv) a polypeptide which comprises at least about 5 contiguous amino acids
of an amino acid sequence encoded by L. intracellularis DNA contained within
a plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B);
NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC);
NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8
ytf N );
(v) a homologue, analogue or derivative of any one of (i) to (iv) which
mimics a B-cell or T-cell epitope of Lawsonia spp.
In an alternative preferred embodiment, the isolated or recombinant
immunogenic
polypeptide is selected from the group consisting of the following:
(i) a polypeptide which comprises an amino acid sequence encoded by a
nucleotide sequence having at least about 60% sequence identity overall to
a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,
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3, 5, 7, 9, 11, 13, 15, and 17;
(ii) a polypeptide which comprises an amino acid sequence encoded by a
nucleotide sequence having at least about 60% sequence identity overall to
the nucleotide sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480
(plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482
(plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a polypeptide encoded by at least about 15 contiguous nucleotides of
a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, and 17;
(iv) a poiypeptide-encoded by at least about 15 contiguous nucleotides of
a nucleotide sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480
(plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482
(plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(v) a homologue, analogue or derivative of any one of (i) to (iv) which
mimics a B-cell or T-cell epitope of Lawsonia spp.
Preferably, the immunogenic polypeptide encompassed by the present invention
has
at least about 70% identity, more preferably at least about 80% identity, even
more
preferably at least about 90% identity, and still even more preferably at
least about
95% identity to the amino acid sequence of a polypeptide selected from the
group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, as
hereinbefore defined.
In determining whether or not two amino acid sequences fall within these
percentage
limits, those skilled in the art will be aware that it is necessary to conduct
a side-by-
side comparison or multiple alignment of sequences. In such comparisons or
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alignments, differences will arise in the positioning of non-identical
residues,
depending upon the algorithm used to perform the alignment. In the present
context,
reference to a percentage sequence identity or similarity between two or more
amino
acid sequences shall be taken to refer to the number of identical and similar
residues
respectively, between said sequences as determined using any standard
algorithm
known to those skilled in the art. For example, amino acid sequence identities
or
similarities may be calculated using the GAP programme of the Computer
Genetics
Group, Inc., University Research Park, Madison, Wisconsin, United States of
America
(Devereaux et al, 1984). The GAP programme utilizes the algorithm of Needleman
and Wunsch (1970) to maximise the number of identical/similar residues and to
minimise the number and/or length of sequence gaps in the alignment.
Alternatively
or in addition, where more than two amino acid sequences are being compared,
the
ClustalW programme of Thompson et al (1994) can be used.
Preferably, the isolated or recombinant immunogenic polypeptide of the
invention
comprises at least about 10 contiguous amino acids of a polypeptide selected
from
the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN
polypeptides, as hereinbefore defined. More preferably, the isolated or
recombinant
immunogenic polypeptide of the invention comprises at least about 20
contiguous
amino acid residues of a polypeptide selected from the group consisting of
flhB, fliR,
ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore
defined.
Even more preferably, the isolated or recombinant immunogenic polypeptide of
the
invention comprises at least about 30 contiguous amino acid residues of a
polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined, and still even
more
preferably, at least about 40 contiguous amino acid residues of said flhB,
fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides.
The present invention further encompasses homologues, analogues and
derivatives
of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA,
motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined.
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"Homologues" of a polypeptide are those immunogenic polypeptides that are
derived
from a full-length L. intracellularis polypeptides described herein, or have
sequence
similarity to a full-length L. intracellularis polypeptide, notwithstanding
one or more
amino acid substitutions, deletions and/or additions relative to the full-
length L.
intracellularis polypeptide. A homologue may also retain the biological
activity or
catalytic activity of the full-length polypeptide. In such homologues, one or
more
amino acids can be replaced by other amino acids having similar properties
such as,
for example, hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity,
propensity to form or break c~helical structures of /3-sheet structures, and
so on.
Substitutional variants are those in which at least one residue in the
sequence has
been removed and a different residue inserted in its place. Amino acid
substitutions
are typically of single residues, but may be clustered depending upon
functional
constraints placed upon the polypeptide; insertions will usually be of the
order of
about 1-10 amino acid residues. and deletions will range from about 1-20
residues.
Preferably, amino acid substitutions will comprise conservative amino acid
substitutions, such as those described supra.
Insertional amino acid sequence variants are those in which one or more amino
acid
residues are introduced into a predetermined site in the protein. Insertions
can
comprise amino-terminal and/or carboxyl terminal fusions as well as intra-
sequence
insertions of single or multiple amino acids. Generally, insertions within the
amino
acid sequence will be smaller than amino or carboxyl terminal fusions, of the
order
of about 1 to 4 residues.
Deletional variants are characterised by the removal of one or more amino
acids from
the sequence.
Amino acid variants of the polypeptide of the present invention may readily be
made
using polypeptide synthetic techniques well known in the art, such as solid
phase
synthesis and the like, or by recombinant DNA manipulations. The manipulation
of
DNA sequences to produce variant proteins which manifest as substitutional,
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insertional or deletional variants are well known in the art. For example,
techniques
for making substitution mutations at predetermined sites in DNA having known
sequence are well known to those skilled in the art, such as by M13
mutagenesis or
other site-directed mutagenesis protocol.
S
"Analogues" are defined as those immunogenic polypeptides that are derived
from
a full-length L. intracellularis polypeptides described herein, or have
sequence
similarity to a full-length L. intracellularis polypeptide, notwithstanding
one or more
non-naturally occurring or modified amino acid residues relative to the
naturally-
occurring full-length L. intracellularis polypeptide. The term "analogue"
shall also be
taken to include an amino acid sequence which is not similar to an amino acid
sequence of a full-length L. intracellularis polypeptide set forth herein,
however
mimics or cross-reacts with a B-cell or T-cell-epitope of Lawsonia spp. and
preferably,
mimics or cross-reacts with a B-cell or T-cell epitope of L. intracellularis,
such as, for
example, a polypeptide which is derived from a computational prediction or
empirical
data revealing the secondary, tertiary or quaternary structure of the full-
length
polypeptide or an epitope thereof.
For example, mimotopes (polypeptide analogues that cross-react with a B-cell
or T-
cell epitope of the Lawsonia polypeptide of the invention but, however,
comprise a
different amino acid sequence to said epitope) may be identified by screening
random amino acid sequences in polypeptide libraries with antibodies that bind
to a
desired T-cell or B-cell epitope. As with techniques for the identification of
B-cell or
T-cell epitopes as described supra, the antibodies used to identify such
mimotopes
may be polyclonal or monoclonal or recombinant antibodies, in crude or
purified form.
Mimotopes of a T-cell epitope may then be assayed further for their ability to
stimulate T-cell cytotoxic or proliferative responses in vitro. Mimotopes are
particularly useful as analogues of non-linear (i.e., conformational) epitopes
of the
polypeptide of the present invention, because conformational epitopes are
generally
formed from non-contiguous regions in a polypeptide, and the mimotopes provide
immunogenic equivalents thereof in the form of a single polypeptide molecule.
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Additionally, the use of polypeptide analogues can result in polypeptides with
increased immunogenic and/or antigenic activity, that are less sensitive to
enzymatic
degradation, and which are more selective. A suitable proline analogue is 2-
aminocyclopentane carboxylic acid (~3AC5c) which has been shown to increase
the
immunogenic activity of a native polypeptide more than 20 times (Mierke et al,
1990;
Portoghese et al, 1990; Goodman et al, 1987).
"Derivatives" of a polypeptide selected from the group consisting of flhB,
fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined,
are
those peptides or polypeptides which comprise at least about five contiguous
amino
acid residues of any one or more of said flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM,
or ytfN polypeptides.
A "derivative" may further comprise additional naturally-occurring, altered
glycosylated, acylated or non-naturally occurring amino acid residues compared
to
the amino acid sequence of a flhB, or fliR, or ntrC, or glnH, or motA, or
motB, or tlyC,
or ytfM, or ytfN polypeptide, as hereinbefore defined. Alternatively or in
addition, a
derivative may comprise one or more non-amino acid substituents such as, for
example, a reporter molecule or other ligand, covalently or non-covalently
bound to
the amino acid sequence of a flhB, or fliR, or ntrC, or glnH, or motA, or
motB, or tlyC,
or ytfM, or ylfN polypeptide, such as, for example, a reporter molecule which
is bound
thereto to facilitate its detection.
Other examples of recombinant or synthetic mutants and derivatives of a
polypeptide
immunogen of the present invention include those incorporating single or
multiple
substitutions in the amino acid sequence of a polypeptide selected from the
group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides.
Recombinant or synthetic mutants and derivatives produced by making deletions
from the amino acid sequence of a polypeptide selected from the group
consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, are
also
included within the scope of preferred derivatives. Additionally, recombinant
or
synthetic mutants and derivatives produced by making additions to a
polypeptide
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selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM,
and ytfN polypeptides, such as, for example, using carbohydrates, lipids
and/or
proteins or polypeptides, are also encompassed by the present invention.
Naturally-occurring or altered glycosylated or acylated forms of the flhB,
fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides are particularly
contemplated by
the present invention.
Additionally, homopolymers or heteropolymers comprising one or more copies of
the
reference polypeptides, or one or more derivatives, homologues or 'analogues
thereof, are clearly within the scope of the present invention.
Preferably, homologues, analogues and derivatives of the flhB, fliR, ntrC,
glni-I, motA,
motB, tlyC, ytfM, or ytfN polypeptides of the invention are "immunogenic",
defined
hereinafter as the ability of said polypeptide, or a derivative, homologue or
analogue
thereof, to elicit B cell and/or T cell responses in the host, in response to
immunization.
Preferred homologues, analogues and derivatives of the flhB, fliR, ntrC, glnH,
motA,
motB, tlyC, ytfM, or ytfN polypeptides of the invention include any amino acid
variant
that functions as B cell or T cell epitope of any one of said polypeptides,
wherein said
variant is capable of mediating an immune response, such as, for example, a
mimotope of the immunogenic polypeptide which has been produced by synthetic
means, such as by Fmoc chemistry. The only requirement of such variant
molecules
is that they cross-react immunologically with a polypeptide selected from the
group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN, as
hereinbefore
defined, or an epitope of said polypeptide.
As will be apparent to those skilled in the art, such homologues, analogues
and
derivatives of the polypeptides of the invention molecules will be useful to
prepare
antibodies that cross-react with antibodies against said polypeptide and/or to
elicit a
protective immune response of similar specificity to that elicited by said
polypeptide.
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Such molecules will also be useful in diagnostic and other applications that
are
immunological in nature such as, for example, diagnostics which utilise one or
more
immunoassay formats (eg. ELISA, RIA and the like).
Accordingly, the immunogen of the present invention or a derivative, homologue
or
analogue thereof is useful in vaccine compositions that protect an individual
against
infection by L. intracellularis and/or as an antigen to elicit polyclonal or
monoclonal
antibody production and/or in the detection of antibodies against L.
intracellularis in
infected animals, particularly in porcine and avian animals.
The polypeptides of the present invention may comprise leader sequences to
facilitate their secretion into the periplasmic space, either as part of the
native protein,
or alternatively, added by recombinant engineering means. Such may have-
improved
immunogenicity compared to non-secreted or non-secretable polypeptides of L.
intracellularis, or non-secreted or non-secretable polypeptides of other
causative
agents of PPE. The particular advantages of such peptides will be immediately
apparent to those skilled in the production of vaccine compositions, where the
inherent immunogenicity of the immunogen is an important consideration for a
protective immune response to be conferred.
Moreover, unique regions of the L. intracellularis polypeptides exemplified
herein are
promising antigenic peptides for the formulation of Lawsonia-specific vaccines
and
diagnostics for the specific detection of Lawsonia spp. in biological samples.
A second aspect of the present invention provides a vaccine composition for
the
prophylaxis or treatment of infection in a mammal or bird by L.
intracellularis or similar
or otherwise related microorganism, said vaccine composition comprising:
(i) an immunogenic component which comprises an isolated or
recombinant polypeptide selected from the group consisting of flhB, fliR,
ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides or an immunogenic
homologue, analogue or derivative of any one of said polypeptides which is
immunologically cross-reactive with L. intracellularis; and
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(ii) one or more carriers, diluents and/or adjuvants suitable for veterinary
or pharmaceutical use.
As used herein, the term "immunogenic component" refers to a polypeptide
encoded
by DNA from, or derived from, L. intracellularis or a related microorganism
thereto
which is capable of inducing a protective immune response in an animal, in
particular
a porcine or avian animal, whether or not said polypeptide is in an isolated
or
recombinant form. Accordingly, the vaccine composition clearly encompasses
those
vaccine compositions which comprise attenuated, killed or non-pathogenic
isolates
or forms of L. intracellularis or related microorganisms thereto which
comprise or
express said polypeptide.
By "protective immune response" is meant that the immunogenic component
elicits
an immune response in the animal-to which the vaccine composition is
administered
at the humoral and/or cellular level which is sufficient to prevent infection
by L.
intracellularis or a related microorganism thereto and/or which is sufficient
to
detectably reduce one or more symptoms or conditions, or to detectably slow
the
onset of one or more symptoms or conditions, associated with infection by L.
intracellularis or a related microorganism thereto in an animal host, as
compared to
a control infected animal. The term "effective amount" of an immunogenic
component present in the vaccine composition refers to that amount of said
immunogenic component that is capable of inducing a protective immune response
after a single complete dose has been administered, or after several divided
doses
have been administered.
Preferably, the polypeptide component of the subject vaccine composition
comprises
an amino acid sequence which is both immunogenic and specific, by virtue of
its
immunological cross-reactivity with the causative agent of PPE, L.
intracellularis. In
this regard, it will be apparent from the preceding description that such
polypeptide
components may comprise the amino acid sequence of a polypeptide of L.
intracellularis as exemplified herein, or alternatively, an immunologically
cross-
reactive homologue, analogue or derivative of said amino acid sequence, such
as,
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for example, a mimotope of said sequence.
The immunogenic polypeptide or immunogenic homologue, analogue or derivative
may be a naturally-occurring polypeptide in isolated or recombinant form
according
to any of the embodiments described supra or exemplified herein. Preferably,
the
immunogenic polypeptide or immunogenic homologue, analogue or derivative is
derived from Lawsonia spp., in particular L. intracellularis or a
microorganism that is
related thereto.
Preferably, the immunogenic component has undergone at least one purification
step
or at least partial concentration from a cell culture comprising L.
intracellularis or a
related microorganism thereto, or from a lysed preparation of L.
intracellularis cells
or related microorganism, or from another culture in which the immunogenic
component is recombinantly expressed. The purity of such a component which has
the requisite immunogenic properties is preferably at least about 20% by
weight of
protein in a particular preparation, more preferably at least about 50%, even
more
preferably at least about 60%, still more preferably at least about 70% and
even more
preferably at least about 80% or greater.
The immunogenic component of the vaccine of the present invention can comprise
a single polypeptide, or a range or combination of different polypeptides
covering
different or similar epitopes. In addition or, alternatively, a single
polypeptide can be
provided with multiple epitopes. The latter type of vaccine is referred to as
a
polyvalent vaccine. A multiple epitope includes two or more epitopes located
within
a polypeptide molecule.
The formulation of vaccines is generally known in the art and reference can
conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack
Publishing Co., Easton, Pennsylvania, USA.
A particularly useful form of the vaccine is a recombinant vaccine produced,
for
example, in a vaccine vector, such as but not limited to a mammalian cell
transfected
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with a vaccinia virus vector, an insect cell transfected with a baculovirus
vector, or a
bacterial cell transfected with a plasmid or cosmid, the only requirement
being that
the vector expresses the immunogenic component.
The present invention clearly extends to recombinant vaccine compositions in
which
the immunogenic component at least is contained within killed vaccine vectors
prepared, for example, by heat, formalin or other chemical treatment, electric
shock
or high or low pressure forces. According to this embodiment, the immunogenic
component of the vaccine is generally synthesized in a live vaccine vector
which is
killed prior to administration to an animal.
Furthermore, the vaccine vector expressing the immunogenic component may be
non-pathogenic or attenuated. Within the scope of this embodiment are cells
that-
have been transfected with non-pathogenic or attenuated viruses encoding the
immunogenic component of the vaccine and non-pathogenic or attenuated cells
that
directly express the immunogenic component.
Attenuated or non-pathogenic host cells include those cells which are not
harmful to
an animal to which the subject vaccine is administered. As will be known to
those
skilled in the art, "live vaccines" can comprise an attenuated virus vector
encoding
the immunogenic component or a host cell comprising same, which is capable of
replicating in an animal to which it is administered, and using host cell
machinery to
express the immunogenic component albeit producing no adverse side-effects
therein. Such vaccine vectors may colonise the gut or other organ of the
vaccinated
animal. Such live vaccine vectors are efficacious by virtue of their ability
to continually
express the immunogenic component in the host animal for a time and at a level
sufficient to confer protective immunity against a pathogen which expresses an
immunogenic equivalent of said immunogenic component. The present invention
clearly encompasses the use of such attenuated or non-pathogenic vectors and
live
vaccine preparations.
The vaccine vector may be a virus, bacterial cell or a eukaryotic cell such as
an
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insect, avian, porcine or other mammalian cell or a yeast cell or a cell line
such as
COS, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby
hamster kidney (BHK) or MDCK cell lines. Suitable prokaryotic cells include
Mycobacterium spp., Corynebacferium spp., Salmonella spp., Escherichia coli,
Bacillus spp. and Pseudomonas spp, amongst others. Bacterial strains which are
suitable for the present purpose are well-known in the relevant art (Ausubel
et al,
1987; Sambrook et al, 1989).
Such cells and cell lines are capable of expression of a genetic sequence
encoding
a polypeptide of the present invention from L. infracellularis, or a
homologue,
analogue or derivative thereof, in a manner effective to induce a protective
immune
response in the animal. For example, a non-pathogenic bacterium can be
prepared
containing an expression vector which comprises-a nucleotide sequence encoding
a polypeptide selected from the group consisting-of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue, or derivative
thereof,
wherein said nucleotide sequence is placed operably under the control of a
constitutive or inducible promoter sequence. The bacterium is then permitted
to
colonise suitable locations in a pig's gut, where it replicates and expresses
the said
polypeptide in amount sufficient to induce a protective immune response
against L.
intracellularis.
In a further alternative embodiment, the vaccine can be a DNA or RNA vaccine
comprising a DNA or RNA molecule encoding a polypeptide selected from the
group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides or
homologues, analogues or derivatives thereof, wherein said vaccine is injected
into
muscular tissue or other suitable tissue in a pig under conditions sufficient
to permit
transient expression of said DNA or RNA to produce an effective amount of said
polypeptide to induce a protective immune response. In a preferred embodiment,
the
DNA vaccine is in the form of a plasmid, in which the DNA is operably
connected with
a promoter region capable of expressing the nucleotide sequence encoding the
immunogen in cells of the immunized animal.
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In the production of a recombinant vaccine, except for a DNA vaccine described
herein, it is therefore necessary to express the immunogenic component in a
suitable
vector system. For the present purpose, the immunogenic component can be
expressed by:
(i) placing an isolated nucleic acid molecule in an expressible format, said
nucleic acid molecule comprising the coding region of a gene selected from
the group consisting of flh8, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN
genes, or a protein-encoding homologue, analogue or derivative thereof;
(ii) introducing the isolated nucleic acid molecule of (i) in an expressible
format into a suitable vaccine vector; and
(iii) incubating or growing the vaccine vector for a time and under
conditions sufficient for expression of the immunogenic component encoded
by said nucleic acid- molecule to occur.
It will be apparent from the preceding discussion that the protein-encoding
region of
a flhB, fliR, ntrC, glnH, motA, mot8, tlyC, ytfM, or ytfN gene comprises a
nucleotide
sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11,
13, 15,
and 17, or alternatively or in addition, a protein-encoding nucleotide
sequence of L.
intracellularis DNA contained within a deposited plasmid selected from the
group
consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH);
NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and
NM01/23286 (plasmid pGTE#8 ytfN).
Preferred homologues of the protein-encoding region of a flhB, fliR, ntrC,
glnH, motA,
motB, tlyC, ytfM, or ytfN gene include those nucleotide sequences selected
from the
group consisting of:
(i) a protein-encoding nucleotide sequence having at least about 60%
sequence identity overall to a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 or a degenerate
variant thereof;
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(ii) a protein-encoding nucleotide sequence having at least about 60%
sequence identity overall to the protein-encoding sequence of L.
intracellularis
DNA contained within a plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid
pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid
pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481
(plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and
NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a protein-encoding nucleotide sequence which comprises at least
about 15 contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
(iv) a protein-encoding nucleotide sequence which comprises at least about
contiguous nucleotides of the protein-encoding sequence of L.
intracellularis DNA contained within a plasmid selected from the group
15 consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH);
NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(v) a protein-encoding nucleotide sequence which hybridizes under at least
low stringency conditions to the complement of a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
and 17; and
(vi) a protein-encoding nucleotide sequence which hybridizes under at least
low stringency conditions to the non-coding strand of L. intracellularis DNA
contained within a plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid
pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid
pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481
(plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and
NM01/23286 (plasmid pGTE#8 ytfN).
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The present invention clearly extends to analogues or derivatives of any one
of (i) to
(vi) which encode a polypeptide which mimics a B-cell or T-cell epitope of
Lawsonia
spp.
For the present purpose, a preferred homologue of the protein-encoding region
of a
flh8, fliR, ntrC, glnH, motA, mot8, tlyC, ytfM, or ytfN gene will have at
least about
80% nucleotide sequence identity to the coding region of said gene, still more
preferably at least about 90% identity, and yet still more preferably at least
about 95%
identity.
In determining whether or not two nucleotide sequences fall within these
percentage
limits, those skilled in the art will be aware that it is necessary to conduct
a side-by-
side comparison or multiple alignment of sequences. In such comparisons or
alignments, differences may arise in the positioning of non-identical
residues,
depending upon the algorithm used to perform the alignment. In the present
context,
reference to a percentage identity between two or more nucleotide sequences
shall
be taken to refer to the number of identical residues between said sequences
as
determined using any standard algorithm known to those skilled in the art. For
example, nucleotide sequences may be aligned and their identity calculated
using the
BESTFIT programme or other appropriate programme of the Computer Genetics
Group, Inc., University Research Park, Madison, Wisconsin, United States of
America
(Devereaux et al, 1984).
Preferably, a homologue of the protein-encoding region of a flh8, fliR, ntrC,
glnH,
motA, mot8, tlyC, ytfM, or ytfN gene hybridizes under at least medium
stringency
conditions to the non-coding strand of said gene, even more preferably under
high
stringency conditions to the non-coding strand of said gene.
For the purposes of defining the level of stringency, a low stringency is
defined herein
as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1 %
(w/v) SDS
at 28°C. A moderate stringency is defined herein as being a
hybridisation and/or
washing carried out in 2xSSC buffer, 0.1 % (w/v) SDS at a temperature in the
range
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45°C to 65°C. A high stringency is defined herein as being a
hybridisation and/or
wash carried out in 0.1 xSSC buffer, 0.1 % (w/v) SDS, or lower salt
concentration, and
at a temperature of at least 65°C. Reference herein to a particular
level of stringency
encompasses equivalent conditions using wash/hybridization solutions other
than
SSC known to those skilled in the art.
Generally, the stringency is increased by reducing the concentration of SSC
buffer,
and/or increasing the concentration of SDS and/or increasing the temperature
of the
hybridisation and/or wash. Those skilled in the art will be aware that the
conditions
for hybridisation and/or wash may vary depending upon the nature of the
hybridisation membrane or the type of hybridisation probe used. Conditions for
hybridisations and washes are well understood by one normally skilled in the
art. For
the purposes of clarification of~the parameters-affectirrg-hybridisation
between nucleic
acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et
al.
(1987), which is herein incorporated by reference.
As used herein, a "nucleic acid molecule in an expressible format" is a
protein-
encoding region of a nucleic acid molecule placed in operable connection with
a
promoter or other regulatory sequence capable of regulating expression in the
vaccine vector system.
Reference herein to a "promoter" is to be taken in its broadest context and
includes
the transcriptional regulatory sequences of a classical genomic gene,
including the
TATA box which is required for accurate transcription initiation, with or
without a
CCAAT box sequence and additional regulatory elements (i.e., upstream
activating
sequences, enhancers and silencers) which alter gene expression in response to
developmental and/or external stimuli, or in a tissue-specific manner. In the
present
context, the term "promoter" is also used to describe a recombinant, synthetic
or
fusion molecule, or derivative which confers, activates or enhances the
expression
of a nucleic acid molecule to which it is operably connected, and which
encodes the
immunogenic polypeptide. Preferred promoters can contain additional copies of
one
or more specific regulatory elements to further enhance expression and/or to
alter the
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spatial expression and/or temporal expression of the said nucleic acid
molecule.
Placing a nucleic acid molecule under the regulatory control of, i.e., "in
operable
connection with", a promoter sequence means positioning said molecule such
that
expression is controlled by the promoter sequence. Promoters are generally,
but not
necessarily, positioned 5' (upstream) to the genes that they control. In the
construction of heterologous promoter/structural gene combinations it is
generally
preferred to position the promoter at a distance from the gene transcription
start site
that is approximately the same as the distance between that promoter and the
gene
it controls in its natural setting, i.e., the gene from which the promoter is
derived.
Furthermore, the regulatory elements comprising a promoter are usually
positioned
within 2 kb of the start site of transcription of the gene. As is known in the
art, some
variation-in this distance can be accommodated without loss of promoter
function.
Similarly, the preferred positioning of a regulatory sequence element with
respect to
a heterologous gene to be placed under its control is defined by the
positioning of the
element in its natural setting, i.e., the genes from which it is derived.
Again, as is
known in the art, some variation in this distance can also occur.
The prerequisite for producing intact polypeptides in bacteria such as E. coli
is the
use of a strong promoter with an effective ribosome binding site. Typical
promoters
suitable for expression in bacterial cells such as E. coli include, but are
not limited to,
the lacz promoter, temperature-sensitive a~ or ~1R promoters, T7 promoter or
the
IPTG-inducible tac promoter. A number of other vector systems for expressing
the
nucleic acid molecule of the invention in E. coli are well-known in the art
and are
described, for example, in Ausubel et al (1987) or Sambrook et al (1989).
Numerous
plasmids with suitable promoter sequences for expression in bacteria and
efficient
ribosome binding sites have been described, such as for example, pKC30 (~,~:
Shimatake and Rosenberg, 1981 ); pKK173-3 (tac: Amann and Brosius, 1985), pET-
3
(T7: Studier and Moffat, 1986); the pBAD/TOPO or pBAD/Thio-TOPO series of
vectors containing an arabinose-inducible promoter (Invitrogen, Carlsbad, CA),
the
latter of which is designed to also produce fusion proteins with thioredoxin
to enhance
solubility of the expressed protein; the pFLEX series of expression vectors
(Pfizer
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Inc., CT, USA); or the pQE series of expression vectors (Qiagen, CA), amongst
others. Typical promoters suitable for expression in viruses of eukaryotic
cells and
eukaryotic cells include the SV40 late promoter, SV40 early promoter and
cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early)
promoter amongst others.
Means for introducing the isolated nucleic acid molecule or a genetic
construct
comprising same into a cell for expression of the immunogenic component of the
vaccine composition are well-known to those skilled in the art. The technique
used
for a given organism depends on the known successful techniques. Means for
introducing recombinant DNA into animal cells include microinjection,
transfection
mediated by DEAE-dextran, transfection mediated by liposomes such as by using
lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-
mediated
DNA uptake, electroporation and microparticle bombardment such as by using DNA-
coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
The immunogenic component of a vaccine composition as contemplated herein
exhibits excellent therapeutic activity, for example, in the treatment and/or
prophylaxis
of PPE when administered in an amount which depends on the particular case.
For
example, for recombinant polypeptide molecules, from about 0.5 ~.g to about 20
mg
may be administered, preferably from about 1 ~g to about 10 mg, more
preferably
from about 10 ~g to about 5 mg, and most preferably from about 50 ~g to about
1 mg
equivalent of the immunogenic component in a volume of about 1m1 to about 5m1.
For DNA vaccines, a preferred amount is from about 0.1 ,ug/ml to about 5 mg/ml
in
a volume of about 1 to about 5 ml. The DNA can be present in "naked" form or
it can
be administered together with an agent facilitating cellular uptake (e.g., in
liposomes
or cationic lipids). The important feature is to administer sufficient
immunogen to
induce a protective immune response. The above amounts can be administered as
stated or calculated per kilogram of body weight. Dosage regime can be
adjusted to
provide the optimum therapeutic response. For example, several divided doses
can
be administered or the dose can be proportionally reduced as indicated by the
exigencies of the therapeutic situation. Booster administration may also be
required.
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The vaccine of the present invention can further comprise one or more
additional
immunomodulatory components such as, for example, an adjuvant or cytokine
molecule, amongst others, that is capable of increasing the immune response
against
the immunogenic component. Non-limiting examples of adjuvants that can be used
in the vaccine of the present invention include the RIBI adjuvant system (Ribi
Inc.,
Hamilton, MT, USA), alum, mineral gels such as aluminium hydroxide gel, oil-in-
water
emulsions, water-in-oil emulsions such as, for example, Block co-polymer
(CytRx,
Atlanta GA, USA),QS-21 (Cambridge Biotech Inc., Cambridge MA, USA), SAF-M
(Chiron, Emeryville CA, USA), AMPHIGEN° adjuvant, Freund's complete
adjuvant;
Freund's incomplete adjuvant; and Saponin, QuilA or other saponin fraction,
monophosphoryl lipid A, and Avridine lipid-amine adjuvant. Other
immunomodulatory
agents that can be included in the vaccine include, for example, one or more
cytokines, such as interferon and/or interleukin, or other known cytokines.
Non-ionic
surfactants such as, for example, polyoxyethylene oleyl ether and n-hexadecyl
polyethylene ether may also be included in the vaccines of the present
invention.
The vaccine composition can be administered in a convenient manner such as by
oral, intravenous (where water soluble), intramuscular, subcutaneous,
intranasal,
intradermal or suppository routes or by implantation (eg., using slow release
technology), provided that a sufficient degree of the immunogenicity of the
immunizing antigen is retained for the purposes of eliciting an immune
response in
the animal being treated. Depending on the route of administration, the
immunogenic
component may be required to be coated in a material to protect it from the
action of
enzymes, acids and other natural conditions which may inactivate it, such as
those
in the digestive tract.
The vaccine composition may also be administered parenterally or
intraperitoneally.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof, or in oils. Under ordinary conditions of storage and use,
these
preparations can contain a preservative to prevent the growth of
microorganisms.
Alternatively, the vaccine composition can be stored in lyophilised form to be
rehydrated with an appropriate vehicle or carrier prior to use.
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Pharmaceutical forms suitable for injectable use include sterile aqueous
solutions
(where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. In all cases the
form must be
fluid to the extent that easy syringability exists, unless the pharmaceutical
form is a
solid or semi-solid such as when slow release technology is employed. In any
event,
it must be stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms.
The carrier may be a solvent or dispersion medium containing, for example,
water,
ethanol, polyol (for example, glycerol, propylene glycol and liquid
polyethylene glycol,
and the like), suitable mixtures thereof and vegetable oils. The proper
fluidity can be
maintained, for example, by the use of a coating such as lecithin, by the
maintenance
of the required particle size in the case of dispersion and by the use of
surfactants.
The prevention of the action of microorganisms can be brought about by various
antibacterial and antifungal agents such as, for example" parabens,
chlorobutanol,
phenol, sorbic acid, thimerosal and the like. In many cases, it will be
preferable to
include isotonic agents such as, for example" sugars or sodium chloride.
Prolonged
absorption of the injectable compositions can be brought about by the use in
the
compositions of agents delaying absorption such as, for example" aluminum
monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound
in the
required amount in the appropriate solvent with various of the other
ingredients
enumerated above, as required, followed by filter-sterilization. Generally,
dispersions
are prepared by incorporating the sterilized active ingredient into a sterile
vehicle
which contains the basic dispersion medium and the required other ingredients
selected from those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods of
preparation are
vacuum drying and the freeze-drying technique which yield a powder of the
active
ingredient plus any additional desired ingredient from previously sterile-
filtered
solution thereof.
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The present invention extends to vaccine compositions which confer protection
against infection by one or more isolates or sub-types of L. intracellularis
including
those that belong to the same serovar or serogroup as L. intracellularis. The
vaccine
composition preferably also confers protection against infection by other
species of
the genus Lawsonia or other microorganisms related thereto, as determined at
the
nucleotide, biochemical, structural, physiological and/or immunointeractive
level; the
only requirement being that said other species or other microorganism
expresses a
polypeptide which is immunologically cross-reactive to a polypeptide selected
from
the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN
polypeptides, or a homologue, analogue or derivative of any one or more of
said
polypeptides as described herein. For example, such related microorganisms may
comprise genomic DNA which is at least about 70% identical overall to the
genomic
DNA of L. intracellularis as determined using standard genomic DNA
hybridisation
and analysis techniques.
The terms "serogroup" and "serovar" relate to a classification of
microorganisms
which is based upon serological typing data, in particular data obtained using
agglutination assays such as the microscopic agglutination test (MAT). Those
skilled
in the art will be aware that serovar and serogroup antigens are a mosaic on
the cell
surface and, as a consequence there will be no strict delineation between
bacteria
belonging to a serovar and/or serogroup. Moreover, organisms which belong to
different species may be classified into the same serovar or serogroup because
they
are indistinguishable by antigenic determination. As used herein, the term
"serovar"
means one or more Lawsonia strains which are antigenically-identical with
respect
to antigenic determinants produced by one or more loci. Quantitatively,
serovars may
be differentiated from one another by cross-agglutination absorption
techniques. As
used herein, the term "serogroup" refers to a group of Lawsonia spp. whose
members cross-agglutinate with shared group antigens and do not cross-
agglutinate
with the members of other groups and, as a consequence, the members of a
serogroup have more or less close antigenic relations with one another by
simple
cross-agglutination.
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The present invention thus clearly extends to vaccine compositions for the
treatment
and/or prophylaxis of animals, in particular, vaccine compositions for the
treatment
and/or prophylaxis of porcine and/or avian species, against any bacterium
belonging
to the same serovar or serogroup as L. intracellularis. Preferably, such
organisms will
express a polypeptide homologue, analogue or derivative of a polypeptide
selected
from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM,
and ytfN
polypeptides.
The present invention extends further to vaccine compositions capable of
conferring
protection against a "genetic variant" of L. intracellularis, the only
requirement being
that said variant expresses a polypeptide which is immunologically cross-
reactive to
a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN polypeptides. Genetic variants of L. intracellularis can
be
developed by mutation, recombination, conjugation or transformation of L.
intracellularis or may occur naturally. It will be known to a person skilled
in the art
how to produce such derivatives.
In a particularly preferred embodiment, the vaccine composition of the
invention is
intended for or suitable for the prophylaxis and/or treatment of infection in
a porcine
or avian animal and more preferably, for prophylaxis and/or treatment of a
porcine
animal for infection by L. intracellularis.
Accordingly, the present invention clearly extends to the use of the
immunogenic
polypeptide of the invention or a DNA or RNA molecule encoding the same,
according to any one of the preceding embodiments or as exemplified herein in
the
preparation of a medicament for the treatment and/or prophylaxis of PPE in
animals,
particularly porcine or avian animals.
The invention further extends to a method of treatment and/or prophylaxis of
PPE in
an animal such as an avian or porcine animal, said method comprising
administering
the vaccine composition or the immunogenic polypeptide of the invention or a
DNA
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or RNA molecule encoding the same, as described or exemplified herein to said
animal for a time and under conditions sufficient for an immune response to
occur
thereto. Preferably, in the case of administration of a vaccine composition,
the
immune response to the immunogen is a protective immune response.
Those skilled in the art will recognise the general applicability of the
invention in
vaccinating animals other than porcine and avian animals against L.
intracellularis
and/or related microorganisms. In the general application of the vaccine of
the
present invention, the only prerequisite is that the animal on which
protection is
conferred is capable of being infected with L. intracellularis and/or a
related
microorganism thereto and that, in the case of a related microorganism to L.
intracellularis, said related microorganism expresses a B-cell or T-cell
epitope which
mimics or cross-reacts with the polypeptide component of the=vaccine
composition
described herein. Animals which may be protected by the vaccine of the present
invention include, but are not limited to, humans, primates, companion animals
(e.g.,
cats, dogs), livestock animals (e.g., pigs, sheep, cattle, horses, donkeys,
goats),
laboratory test animals (e.g., mice, rats, guinea pigs, rabbits) and captive
wild animals
(e.g., kangaroos, foxes, deer). The present invention also extends to the
vaccination
of birds such as poultry birds, game birds and caged birds.
The present invention further extends to combination vaccines comprising an
effective amount of a first immunogenic component comprising a polypeptide
selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM,
and ytfN polypeptides, or a homologue, analogue or derivative thereof as
described
herein, or a DNA or RNA molecule encoding the same, combined with an effective
amount of a second immunogenic component comprising one or more other antigens
capable of protecting a porcine animal, or bird, against either Lawsonia spp.
or
another pathogen that infects and causes disease in said animal. The second
immunogenic component is different from the first immunogenic component and is
preferably selected from the group consisting of the L. intracellularis FIgE,
hemolysin,
OmpH, SodC, flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides
and homologues, analogues or derivatives thereof. The present invention
clearly
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extends to DNA vaccines and vaccine vectors which express said first
immunogenic
component and said second immunogenic component.
It is within the scope of the invention to encompass vaccine compositions
comprising
multimeric and polymeric forms of any one or more of the immunogenic
polypeptides
described herein, such as tandem arrays of homologous amino acid sequences,
or,
alternatively, tandem arrays of heterologous immunogenic repeats of amino acid
sequences. The present invention extends further to nucleic acid molecules
encoding
such polymeric forms.
The isolated or recombinant polypeptide of the invention, or an
immunologically-
equivalent homologue, analogue or derivative thereof is also useful for the
preparation of immunologically -interactive molecules which are useful in the
diagnosis of infection-of an-animal by Lawsonia spp., in particular by L.
intracellularis
or a related organism thereto.
As used herein, the term "immunologically interactive molecule" includes
antibodies
and antibody derivatives and functional equivalents, such as a Fab, or a SCAB
(single-chain antibody), any of which optionally can be conjugated to an
enzyme,
radioactive or fluorescent tag, amongst others. The only requirement of such
immunologically interactive molecules is that they are capable of binding
specifically
to the immunogenic polypeptide of the present invention as hereinbefore
described.
Accordingly, a further aspect of the invention extends to an immunologically
interactive molecule which is capable of binding to a polypeptide selected
from the
group consisting of:
(i) a polypeptide which comprises an amino acid sequence which has at
least about 60% sequence identity overall to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(ii) a polypeptide which comprises an amino acid sequence which has at
least about 60% sequence identity overall to an amino acid sequence
encoded by L. intracellularis DNA contained within a plasmid selected from the
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group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a polypeptide which comprises at least about 5 contiguous amino acids
of an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(iv) a polypeptide which comprises at least about 5 contiguous amino acids
of an amino acid sequence encoded by L. intracellularis DNA contained within
a plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B);
NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC);
NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8
y~N );
(v) a polypeptide which comprises an amino acid sequence encoded by a
nucleotide sequence having at least about 60% sequence identity overall to
a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, and 17;
(vi) a polypeptide which comprises an amino acid sequence encoded by a
nucleotide sequence having at least about 60% sequence identity overall to
the nucleotide sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480
(plasmid .pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482
(plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(vii) a polypeptide encoded by at least about 15 contiguous nucleotides of
a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, and 17;
(viii) a polypeptide encoded by at least about 15 contiguous nucleotides of
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a nucleotide sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480
(plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482
(plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and
(ix) a homologue, analogue or derivative of any one of (i) to (viii) which
mimics a B-cell or T-cell epitope of Lawsonia spp.
In a preferred embodiment, the immunologically interactive molecule is an
antibody
that binds specifically to one or more epitopes of a polypeptide selected from
the
group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides. More preferably, the immunologically interactive molecule binds
specifically to one or more epitopes of a polypeptide from a causative agent
of PPE,
such as, for example, L. infracellularis.
Conventional methods can be used to prepare the immunologically interactive
molecules. For example, by using a polypeptide immunogen of the present
invention,
polyclonal antisera or monoclonal antibodies can be made using standard
methods.
For example, a mammal, (e.g., a mouse, hamster, or rabbit) can be immunized
with
an immunogenic form of the polypeptide of the present invention which elicits
an
antibody response in the mammal. Techniques for conferring immunogenicity on a
polypeptide include conjugation to carriers, or other techniques well known in
the art.
For example, the polypeptide can be administered in the presence of adjuvant
or can
be coupled to a carrier molecule, as known in the art, that enhances the
immunogenicity of the polypeptide. The progress of immunization can be
monitored
by detection of antibody titres in plasma or serum. Standard ELISA or other
immunoassay can be used with the immunogen as antigen to assess the levels of
antibodies. Following immunization, antisera can be obtained and, for example,
IgG
molecules corresponding to the polyclonal antibodies can be isolated from the
antisera.
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To produce monoclonal antibodies, antibody producing cells (lymphocytes) can
be
harvested from an animal immunised with a polypeptide of the present invention
and
fused with myeloma cells by standard somatic cell fusion procedures, thus
immortalizing these cells and yielding hybridoma cells. Such techniques are
well
known in the art, for example, the hybridoma technique originally developed by
Kohler and Milstein (1975), as well as other techniques such as the human B-
cell
hybridoma technique (Kozbor et al., 1983), the EBV-hybridoma technique to
produce
human monoclonal antibodies (Cole et al., 1985), and screening of
combinatorial
antibody libraries (Huse et al., 1989). Hybridoma cells can be isolated and
screened
immunochemically for production of antibodies that are specifically reactive
with the
polypeptide and monoclonal antibodies isolated therefrom.
As with all immunogenic compositions for eliciting antibodies; the
immunogenically
effective amounts of the peptides of the invention must-be determined
empirically.
Factors to be considered include the immunogenicity of the native polypeptide,
whether or not the polypeptide will be complexed with or covalently attached
to an
adjuvant or carrier protein or other carrier, the route of administration for
the
composition, i.e., intravenous, intramuscular, subcutaneous, etc., and the
number of
immunizing doses to be administered. Such factors are known in the vaccine art
and
it is well within the skill of immunologists to make such determinations
without undue
experimentation.
The term "antibody" as used herein, is intended to include fragments thereof
which
are also specifically reactive with a polypeptide that mimics or cross-reacts
with a B-
cell or T-cell epitope of the L. intracellularis polypeptide selected from the
group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides.
Antibodies can be fragmented using conventional techniques and the fragments
screened for utility in the same manner as described above for whole
antibodies. For
example, F(ab')2 fragments can be generated by treating antibody with pepsin.
The
resulting F(ab')2 fragment can be treated to reduce disulfide bridges to
produce Fab'
fragments.
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It is within the scope of this invention to include any secondary antibodies
(monoclonal, polyclonal or fragments of antibodies), including anti-idiotypic
antibodies, directed to the first mentioned antibodies discussed above. Both
the first
and second antibodies can be used in detection assays or a first antibody can
be
used with a commercially available anti-immunoglobulin antibody. An antibody
as
contemplated herein includes any antibody specific to any region of a
polypeptide
which mimics, or cross-reacts with a B-cell or T-cell epitope of a L.
intracellularis
polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN polypeptides.
The antibodies described herein are useful for determining B-cell or T-cell
epitopes
of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA,
motB, tlyC, ytfM, and ytfN polypeptides, such as, for example, by testing the
ability
of synthetic peptides to cross-react immunologically with said polypeptide or
to elicit
the production of antibodies which cross-react with said polypeptide. Using
methods
described herein, polyclonal antibodies, monoclonal antibodies or chimeric
monoclonal antibodies can also be raised to peptides which mimic or cross-
react with
a B-cell or T-cell epitope of a L. intracellularis polypeptide selected from
the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides.
More particularly, the polyclonal, monoclonal or chimeric monoclonal
antibodies can
be used to detect the polypeptide of the invention and/or any homologues,
analogues
or derivatives thereof, in various biological materials. For example, they can
be used
in an ELISA, radioimmunoassay, or histochemical test. In other words, the
antibodies
can be used to test for binding to a polypeptide of the invention or to a
homologue,
analogue or derivative thereof, in a biological sample to diagnose the
presence of L.
intracellularis therein.
Accordingly, a further aspect of the invention provides a method of diagnosing
infection of an animal by L. intracellularis or a related microorganism
thereto, said
method comprising the steps of contacting a biological sample derived from
said
animal with an immunologically interactive molecule which is capable of
binding to
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a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or derivative
thereof, for
a time and under conditions sufficient for an antigen:antibody complex to
form, and
detecting said complex formation.
According to this embodiment of the present invention, the immunologically
interactive molecule is preferably an antibody molecule prepared against a L.
intracellularis polypeptide selected from the group consisting of flhB, fliR,
ntrC, glnH,
motA, motB, tlyC, ytfM, and ytfN polypeptides, or an analogue or derivative
thereof.
If the biological sample being tested contains one or more epitopes of a
polypeptide
selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM,
and ytfN polypeptides, or an immunologically cross-reactive homologue,
analogue
or derivative thereof, it will give a positive binding result to the
immunologically
interactive molecule.
Preferably, the biological sample is derived from a porcine or avian host of
the
pathogen L. intracellularis or a related microorganism thereto, and includes
an
appropriate tissue or fluid sample from the animal.
Preferred biological samples are derived from the ileum, caecum, small
intestine,
large intestine, whole serum or lymph nodes of the porcine or avian host
animal being
tested. Alternatively or in addition the biological test sample may comprise
faeces
or a rectal swab derived from the animal.
To distinguish L. intracellularis from other microorganisms resident in the
gut or other
organ of an animal, the antibodies should not be prepared against highly-
conserved
epitopes of the L. intracellularis polypeptide, such as, for example, those
amino acid
sequences of at least 5 amino acids in length which are conserved between L.
infracellularis and a microorganism which is present in the gut or other organ
of an
animal in respect of which diagnosis is sought such as, for example, E.coli.
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Conventional immunoassays can be used to perform this embodiment of the
invention. A wide range of immunoassay techniques are available as can be seen
by
reference to US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of
course,
include both single-site and two-site or "sandwich" assays of the non-
competitive
types, as well as the traditional competitive binding assays. These assays
also
include direct binding of a labelled antibody to a target. It will be readily
apparent to
the skilled technician how to modify or optimise such assays to perform this
embodiment of the present invention, and all such modifications and
optimisations
are encompassed by the present invention.
In one alternative embodiment, the present invention contemplates a method of
identifying whether or not an animal has suffered from a past infection, or is
currently
infected with L. intracellularis or a related microorganism thereto, said
method-
comprising contacting blood or serum derived from said animal with the
immunogenic
polypeptide of the invention for a time and under conditions sufficient for an
antigen:antibody complex to form, and detecting said complex formation. This
embodiment differs from the embodiment described supra in that it relies upon
the
detection of circulating antibodies against L. intracellularis or related
organism in the
animals blood or serum which are present as a consequence of a past or present
infection by this pathogen. However, it will be apparent to those skilled in
the art that
the principle of the assay format is the same. As with other embodiments of
the
invention referred to supra, conventional immunoassays can be used. Persons
skilled
in the art will readily be capable of varying known immunoassay formats to
perform
the present embodiment. This embodiment of the invention can also utilise
derivatives of blood and serum which comprise immunologically interactive
molecules
such as, for example, partially-purified IgG or IgM fractions and huffy coat
samples,
amongst others. The preparation of such fractions will also be known to those
skilled
in the art.
A further aspect of the present invention provides an isolated nucleic acid
molecule
which comprises a sequence of nucleotides that encodes, or is complementary to
a
nucleic acid molecule that encodes a polypeptide selected from the group
consisting
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of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides,
including any
and all genes selected from the group consisting of flh8, fliR, ntrC, glnH,
motA, mot8,
flyC, ytfM, and ytfN genes as defined hereinabove.
In a preferred embodiment, the isolated nucleic acid molecule comprises a
nucleotide
sequence encoding a polypeptide that is immunologically cross-reactive with L.
intracellularis or other causative agent of PPE, wherein said nucleotide
sequence is
selected from the group consisting of:
(i) a nucleotide sequence having at least about 60% sequence identity
overall to a nucleotide sequence selected from the group consisting of SEQ
I D NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
(ii) a nucleotide sequence having at least about 60% sequence identity
overall to L. intracellularis DNA contained within-a plasmid selected from the
group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(iii) a nucleotide sequence which comprises at least about 15 contiguous
nucleotides of a sequence selected from the group consisting of SEQ ID NOs:
1, 3, 5, 7, 9, 11, 13, 15, and 17;
(iv) a nucleotide sequence which comprises at least about 15 contiguous
nucleotides of L. intracellularis DNA contained within a plasmid selected from
the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
(v) a nucleotide sequence which hybridizes under at least low stringency
conditions to a nucleotide sequence selected from the group consisting of
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 or a complementary nucleotide
sequence thereto;
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(vi) a nucleotide sequence which hybridizes under at least low stringency
conditions to L. intracellularis DNA contained within a plasmid selected from
the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and
(vii) a homologue, analogue or derivative of any one of (i) to (vi) which
encodes a polypeptide which mimics a B-cell or T-cell epitope of Lawsonia
spp.
For the present purpose, a "homologue" of a nucleotide sequence shall be taken
to
refer to an isotated nucleic acid molecule which encodes a polypeptide that is
immunologically cross-reactive to a polypeptide selected from the group
consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, but
which
includes one or more nucleotide substitutions, insertions, deletions, or
rearrangements.
An "analogue" of a nucleotide sequence set forth herein shall be taken to
refer to an
isolated nucleic acid molecule which encodes a polypeptide which is
immunologically
cross-reactive to a polypeptide selected from the group consisting of flhB,
fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, but which includes one or
more
non-nucleotide constituents not normally present in said isolated nucleic acid
molecule, such as, for example, carbohydrates, radiochemicals including radio
nucleotides, reporter molecules such as, but not limited to biotin, DIG,
alkaline
phosphatase or horseradish peroxidase, amongst others.
A "derivative" of a nucleotide sequence set forth herein shall be taken to
refer to any
isolated nucleic acid molecule which contains at least about 60% nucleotide
sequence identity to 15 or more contiguous nucleotides present in the
nucleotide
sequence of a gene selected from the group consisting of flh8, fliR, ntrC,
glnH, motA,
mot8, tlyC, ytfM, and ytfN genes.
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Generally, a flh8, fliR, ntrC, glnH, motA, mot8, tlyC, ytfM, or ytfN gene may
be
subjected to mutagenesis to produce single or multiple nucleotide
substitutions,
deletions and/or insertions. Nucleotide insertional derivatives include 5' and
3~
terminal fusions as well as intra-sequence insertions of single or multiple
nucleotides
or nucleotide analogues. Insertional nucleotide sequence variants are those in
which
one or more nucleotides or nucleotide analogues are introduced into a
predetermined
site in the nucleotide sequence of the gene, although random insertion is also
possible with suitable screening of the resulting product being performed.
Deletional
nucleotide sequence variants are characterised by the removal of one or more
nucleotides from the gene. Substitutional nucleotide sequence variants are
those in
which at least one nucleotide in the gene sequence has been removed and a
different nucleotide or nucleotide analogue inserted in its place. In a
preferred
embodiment, such substitutions are selected based on the degeneracy of the
genetic
code, as known in the art, with the resulting substitutional variant encoding
the amino
acid sequence of a flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN
polypeptide.
Preferred homologues, analogues and derivatives of a flh8, fliR, ntrC, glnH,
motA,
mot8, tlyC, ytfM, or ytfN gene comprise a sequence of nucleotides which has at
least
about 80%identity, even more preferably at least about 90% identity, and yet
still
more preferably at least about 95% identity to said gene.
In determining whether or not two nucleotide sequences fall within these
percentage
limits, reference is made to the description supra of methods for conducting a
side-
by-side comparison or multiple alignment of nucleotide sequences.
Alternatively or in addition, preferred homologues, analogues and derivatives
of a
flh8, fliR, ntrC, glnH, motA, mot8, tlyC, ytfM, or ytfN gene comprise a
sequence of
nucleotides which hybridizes under at least moderate stringency conditions and
to the
nucleotide sequence of said gene, or to a nucleic acid fragment comprising at
least
about 20 contiguous nucleotides in length derived therefrom, and even more
preferably, under high stringency conditions to said gene, or to said nucleic
acid
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fragment. For the purposes of defining the level of stringency, reference is
made to
the description hereinabove of hybridization stringencies.
In a more preferred embodiment, such a nucleotide sequence encodes a
polypeptide
that is immunologically cross-reactive with L. intracellularis or other
causative agent
of PPE.
In a particularly preferred embodiment, the isolated nucleic acid molecule of
the
present invention comprises or consists of a nucleotide sequence selected from
the
group consisting of:
(i) a nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1,3, 5, 7, 9, 11, 13, 15, and 17;
(ii) a nucleotide sequence of the L. intracellularis DNA contained within a
deposited plasmid selected from the group consisting of AGAL Accession
Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2
flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
(iii) a nucleotide sequence that encodes the same polypeptide as (i) or (ii),
wherein said polypeptide is selected from the group consisting of flhB, fliR,
ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides; and
(iv) a nucleotide sequence that is complementary to (i) or (ii) or (iii).
The present invention clearly encompasses genetic constructs comprising the
subject
nucleic acid molecule in an expressible format suitable for the preparation of
a
recombinant immunogenic polypeptide selected from the group consisting of
flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, such as for
use in
recombinant univalent or polyvalent recombinant vaccines.
In such cases, the nucleic acid molecule will be operably connected to a
promoter
sequence which can thereby regulate expression of said nucleic acid molecule
in a
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prokaryotic or eukaryotic cell as described supra.
The genetic construct optionally further comprises a terminator sequence. The
term
"terminator" refers to a DNA sequence at the end of a transcriptional unit
which
signals termination of transcription. A "terminator" is a nucleotide sequence,
generally located within the 3'-non-translated region of a gene or mRNA,
comprising
a polyadenylation signal to facilitate the post-transcriptional addition of a
polyadenylate sequence to the 3'-end of a primary mRNA transcript. Terminator
sequences may be isolated from the genetic sequences of bacteria, fungi,
viruses,
animals and/or plants. Terminators active in animal cells are known and
described
in the literature.
In a preferred embodime~.t, the genetic construct can be a cloning or
expression
vector, as known in the art, such as a plasmid, cosmid, or phage, comprising a
nucleic acid molecule of the present invention, and host cells transformed or
transfected therewith. In a non-limiting embodiment, the vector is a plasmid
selected
from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and
NM01/23286 (plasmid pGTE#8 ytfN).
The genetic constructs of the present invention are particularly useful for
producing
the immunogenic component of the vaccine composition described herein or for
use
in a DNA vaccine.
A range of genetic diagnostic assays to detect infection of an animal by L.
intracellularis or a related microorganism can be employed using the nucleic
acid
molecule described herein such as, for example, assays based upon the
polymerise
chain reaction (PCR) and nucleic acid hybridisation. All such assays are
contemplated in the present invention.
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Accordingly, a still further aspect of the invention provides a diagnostic
method of
detecting L. infracellularis or related microorganism in a biological sample
derived
from an animal subject, said method comprising the steps of hybridising one or
more
probes or primers derived from a nucleotide sequence of a flhB, fliR, ntrC,
glnH,
motA, motB, tlyC, ytfM, or yffN gene as defined hereinabove, or a homologue,
analogue or derivative thereof, to a DNA or RNA molecule present in said
sample
and then detecting said hybridisation using a detection means.
As used herein, the term "probe" refers to a nucleic acid molecule which is
capable
of being used in the detection of a gene selected from the group consisting of
flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes. Probes may comprise
DNA
(single-stranded or double-stranded) or RNA (i.e., riboprobes) or analogues
thereof.
The term "primer" refers to a probe as hereinbefore defined which is further
capable
of being used to amplify a nucleotide sequence from L. intracellularis or a
related
microorganism thereto in a PCR.
Preferred probes and primers include fragments of a gene selected from the
group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes,
including
synthetic single-stranded DNA or RNA molecules of at least about 15
nucleotides in
length.
Preferably, probes and primers according to this embodiment will comprise at
least
about 20 contiguous nucleotides in length from a gene selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes,
even more
preferably at least about 25 contiguous nucleotides, still even more
preferably at least
about 50 contiguous nucleotides, and even more preferably at least about 100
nucleotides to about 500 nucleotides in length from said gene. Probes and
primers
comprising the full-length gene or a complementary nucleotide sequence thereto
are
also encompassed by the present invention.
Probes or primers can comprise inosine, adenine, guanine, thymidine, cytidine
or
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uracil residues or functional analogues or derivatives thereof that are
capable of
being incorporated into a polynucleotide molecule, provided that the resulting
probe
or primer is capable of hybridising under at least low stringency conditions
to a gene
selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM,
and yffN genes, or is at least about 60% identical to one strand of said gene.
The biological sample according to this aspect of the invention includes any
organ,
tissue, cell or exudate which contains or is likely to contain L.
intracellularis or a
nucleic acid derived therefrom. A biological sample can be prepared in a
suitable
solution such as, for example, an extraction buffer or suspension buffer. The
present
invention extends to the testing of biological solutions thus prepared, the
only
requirement being that said solution at least comprises a biological sample as
described herein.
The diagnostic assay of the present invention is useful for the detection of
L.
intracellularis or a microorganism which is related thereto which expresses a
polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB,
tlyC, ytfM, and ytfN polypeptides.
The present invention clearly contemplates diagnostic assays which are capable
of
both genus-specific and species-specific detection. Accordingly, in one
embodiment,
the probe or primer, or a homologue, analogue or derivative thereof, comprises
DNA
capable of being used to detect multiple Lawsonia spp. In an alternative
embodiment,
the probe or primer or a homologue, analogue or derivative thereof comprises
DNA
capable of being used to distinguish L. intracellularis from related
microorganisms.
Less-highly conserved regions within the flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM,
or ytfN genes are particularly useful as species-specific probes and/or
primers for the
detection of L. intracellularis and very closely related species.
Furthermore, the diagnostic assays described herein can be adapted to a genus-
specific or species-specific assay by varying the stringency of the
hybridisation step.
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Accordingly, a low stringency hybridisation can be used to detect several
different
species of Lawsonia in one or more biological samples being assayed, while a
high
stringency hybridisation can be used to distinguish L, intracellularis from
such other
species.
The detection means according to this aspect of the invention may be any
nucleic
acid-based detection means such as, for example, nucleic acid hybridisation
techniques or paper chromatography hybridisation assay (PACHA), or an
amplification reaction such as PCR, or nucleic acid sequence-based
amplification
(NASBA) system. The invention further encompasses the use of different assay
formats of said nucleic acid-based detection means, including restriction
fragment
length polymorphism (RFLP), amplified fragment length polymorphism (AFLP),
single-strand chain polymorphism (SSCP), amplification and mismatch detection
(AMD), interspersed repetitive sequence polymerise chain reaction (IRS-PCR),
inverse polymerise chain reaction (iPCR), in situ polymerise chain reaction
and
reverse transcription polymerise chain reaction (RT-PCR), amongst others.
Where the detection means is a nucleic acid hybridisation technique, the probe
can
be labelled with a reporter molecule capable of producing an identifiable
signal (e.g.,
a radioisotope such as 32P Or 35S, or a biotinylated molecule). According to
this
embodiment, those skilled in the art will be aware that the detection of said
reporter
molecule provides for identification of the probe and that, following the
hybridisation
reaction, the detection of the corresponding nucleotide sequences in the
biological
sample is facilitated. Additional probes can be used to confirm the assay
results
obtained using a single probe.
A variation of the nucleic acid hybridisation technique contemplated by the
present
invention is the paper chromatography hybridisation assay (PACHA) described by
Reinhartz et al. (1993) and equivalents thereof, wherein a target nucleic acid
molecule is labelled with a reporter molecule such as biotin, applied to one
end of a
nitrocellulose or nylon membrane filter strip and subjected to chromatography
under
the action of capillary or other forces (e.g., an electric field) for a time
and under
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conditions sufficient to promote migration of said target nucleic acid along
the length
of said membrane to a zone at which a DNA probe is immobilised thereto such
as,
for example, in the middle region. According to this detection format,
labelled target
nucleic acid comprising the Lawsonia spp. nucleotide sequences complementary
to
the probe will hybridise thereto and become immobilised in that region of the
membrane to which the probe is bound. Non-complementary sequences to the probe
will diffuse past the site at which the probe is bound. The target nucleic
acid may
comprise a crude or partially-pure extract of DNA or RNA or, alternatively, an
amplified or purified DNA. Additional variations of this detection means which
utilise
the nucleotide sequences described herein are clearly encompassed by the
present
invention.
Wherein the detection-mEans is a RFLP, nucleic acid derived from the
biological
sample, in particular DNA, is digested with one or more restriction
endonuclease
enzymes and the digested DNA is subjected to electrophoresis, transferred to a
solid
support such as, for example, a nylon or nitrocellulose membrane, and
hybridised to
a probe optionally labelled with a reporter molecule as hereinbefore defined.
According to this embodiment, a specific pattern of DNA fragments is displayed
on
the support, wherein said pattern is preferably specific for a particular
Lawsonia spp.,
to enable the user to distinguish between different species of the bacterium.
Wherein the detection means is an amplification reaction such as, for example,
a
polymerise chain reaction or a nucleic acid sequence-based amplification
(NASBA)
system or a variant thereof, one or more nucleic acid primer molecules of at
least 15
contiguous nucleotides in length derivable from a gene selected from the group
consisting of flh8, fliR, ntrC, glnH, motA, mot8, tlyC, ytfM, and ytfN genes
is
hybridised to nucleic acid derived from a biological sample, and nucleic acid
copies
of the FIgE-encoding genetic sequences in said sample, or a part or fragment
thereof, are enzymically-amplified.
Those skilled in the art will be aware that there must be a sufficiently high
percentage
of nucleotide sequence identity between the primers and the sequences in the
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biological sample template molecule to which they hybridise (i.e., the
"template
molecule"). As stated previously, the stringency conditions can be selected to
promote hybridisation.
Preferably, each primer is at least about 95% identical to a region of a gene
selected
from the group consisting of flh8, fliR, nfrC, glnH, motA, mot8, tlyC, ytfM,
and ytfN
genes in the template molecule to which it hybridises.
Those skilled in the art will also be aware that, in one format, PCR provides
for the
hybridisation of non-complementary primers to different strands of the
template
molecule, such that the hybridised primers are positioned to facilitate the
5'~ 3'
synthesis of nucleic acid in the intervening region, under the control of a
thermostable
DNA polymerase enzyme. As a consequence, PCR provides an advantage over
other detection means in so far as the nucleotide sequence in the region
between the
hybridised primers may be unknown and unrelated to any known nucleotide
sequence.
In an alternative embodiment, wherein the detection means is AFLP, the primers
are
selected such that, when nucleic acid derived from the biological sample, in
particular
DNA, is amplified, different length amplification products are produced from
different
Lawsonia spp. The amplification products can be subjected to electrophoresis,
transferred to a solid support such as, for example, a nylon or nitrocellulose
membrane, and hybridised to a probe optionally labelled with a reporter
molecule as
hereinbefore described. According to this embodiment, a specific pattern of
amplified
DNA fragments is displayed on the support, said pattern optionally specific
for a
particular Lawsonia ssp., to enable the user to distinguish between different
species
of the bacterium in much the same way as for RFLP analysis.
The technique of AMD facilitates, not only the detection of Lawsonia spp. DNA
in a
biological sample, but also the determination of nucleotide sequence variants
which
differ from the primers and probes used in the assay format. Wherein the
detection
means is AMD, the probe is end-labelled with a suitable reporter molecule and
mixed
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with an excess of the amplified template molecule. The mixtures are
subsequently
denatured and allowed to renature to form nucleic acid "probeaemplate hybrid
molecules" or "hybrids", such that any nucleotide sequence variation between
the
probe and the temple molecule to which it is hybridised will disrupt base-
pairing in the
hybrids. These regions of mismatch are sensitive to specific chemical
modification
using hydroxylamine (mismatched cytosine residues) or osmium tetroxide
(mismatched thymidine residues), allowing subsequent cleavage of the modified
site
using piperidine. The cleaved nucleic acid may be analysed using denaturing
polyacrylamide gel electrophoresis, followed by standard nucleic acid
hybridisation
as described supra, to detect the Lawsonia-derived nucleotide sequences. Those
skilled in the art will be aware of the means of end-labelling a genetic probe
according to the performance of the invention described in this embodiment.
According to this embodiment, the use of a single end-labelled probe allows
unequivocal localisation of the sequence variation. The distance between the
points)
of sequence variation and the end-label is represented by the size of the
cleavage
product.
In an alternative embodiment of AMD, the probe is labelled at both ends with a
reporter molecule, to facilitate the simultaneous analysis of both DNA
strands.
Wherein the detection means is RT-PCR, the nucleic acid sample comprises an
RNA
molecule which is a transcription product of Lawsonia-derived DNA or a
homologue,
analogue or derivative thereof. As a consequence, this assay format is
particularly
useful when it is desirable to determine expression of one or more Lawsonia
genes.
According to this embodiment, the RNA sample is reverse-transcribed to produce
the complementary single-stranded DNA which is subsequently amplified using
standard procedures.
Variations of the embodiments described herein are described in detail by
McPherson et al. (1991 ).
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The present invention clearly extends to the use. of any and all detection
means
referred to supra for the purposes of diagnosing Lawsonia spp. and in
particular L.
intracellularis infection in animals.
The amplification reaction detection means described supra can be further
coupled
to a classical hybridisation reaction detection means to further enhance
sensitivity
and specificity of the inventive method, such as by hybridising the amplified
DNA with
a probe which is different from any of the primers used in the amplification
reaction.
Similarly, the hybridisation reaction detection means described supra can be
further
coupled to a second hybridisation step employing a probe which is different
from the
probe used in the first hybridisation reaction.
A further aspect of the invention provides an isolated probe or primer derived
from
a gene selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC,
ytfM, and ytfN genes. Preferably, the probe or primer of the invention
comprises a
nucleotide sequence selected from the group consisting of SEQ ID NO: 19 to SEQ
ID NO: 68 or a complementary nucleotide sequence thereto.
The present invention does not extend to any nucleic acid or polypeptide of
Camplylobacteror Helicobacterthat was disclosed publicly before the filing
date or
priority date of this application, or otherwise takes priority over the
instant application,
and which is homologous to a nucleotide sequence or amino acid sequence of
Lawsonia spp. disclosed herein.
The present invention is further described with reference to the following non-
limiting
Examples.
EXAMPLE 1
Molecular Cloning of Lawsonia intracellularis genes
Isolation of DNA and construction of DNA libraries
L. intracellularis DNA was purified from pig intestinal mucosa isolated from
the ileum
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of pigs experimentally infected with L. intracellularis. DNA purification from
homogenized intestinal mucosa was performed according to the method of Nollau
et
al. (1996); or alternatively, by phenol extraction and sodium acetate-ethanol
precipitation of DNA.
To facilitate cloning of L. intracellularis gene sequences, several genomic
libraries
were constructed. These libraries were specifically modified by ligation of a
known
sequence (Vectorette IIT"", Genosys Biotechnologies, Inc., The Woodlands, TX)
to
the 5'- and 3'- ends of restricted DNA fragments. VectoretteT"' libraries were
constructed by separately digesting aliquots of L. intracellularis-infected
pig mucosal
DNA extract with restriction endonucleases Hindlll, EcoRl, Dral or Hpal at
37°C
overnight. The reaction was then spiked with additional fresh restriction
enzyme and
adjusted to 2 mM ATP, 2 mM DTT final concentration. VectoretteT"' tailing was
carried out by addition of T4 DNA Ligase (1 unit) plus 3 pMol of the
appropriate
compatible VectoretteT"" linker (Hindlll VectoretteT"~: Hindlll-digested DNA;
EcoRl:
EcoRl digested DNA; Blunt: Dral-, Hpal- digested DNA). The mixture was
incubated
for three cycles, each cycle consisting of 20°C for 60 min; followed by
37°C for 30
min, to complete the tailing reaction. Reaction volumes were then adjusted to
200 ~.I
with water, and reactions were stored at -20°C.
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EXAMPLE 2
Expression of the YtfN and YtfM genes of L, intracellularis
i) Isolation of a C-terminal fragment of ytfN gene by genome walking
The complete sequence of the L. intracellularis YtfN gene was determined from
genomic DNA and is set forth herein as,SEQ ID NO: 17. Based upon the 2,035 by
sequence obtained for the amino terminal portion of the ytfN gene fragment,
oligonucleotide primer KWK-Li-YtfN-4C (SEQ ID N0:29) was designed and
synthesized (Life Technologies; Rockville, MD). This oligonucleotide binds
within the
3'-region of the YtfN gene in the L. intracellularis chromosome to allow
amplification
of DNA downstream of the existing gene fragment. For polymerase chain
amplification, primer KWK-Li-YtfN-4C (SEQ ID N0:29) was used in combination
with
a VectoretteT"" specific oligonucleotide primer (ER70; SEQ ID N0:108) in 50 ~I
reactions containing 1 x PCR Buffer II (Perkin Elmer; Foster City, CA), 2.0 mM
MgCl2,
250 ~,M each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpIiTaqT"" Gold
(Perkin
Elmer) thermostable polymerase. Reactions were performed with 1 ~,I of the
VectoretteT"" libraries as DNA template. Amplification was carried out as
follows:
denaturation (94°C, 9 min); 40 cycles of denaturation (94°C, 30
sec), annealing
(60°C, 30 sec), and polymerization (72°C, 4.0 min); this was
followed by a final
extension at 72°C for 7 minutes.
The amplified products were visualized by separation on a 1.0% agarose gel
(Sigma;
St. Louis, Mo). Screening of the Hpal library by PCR resulted in amplification
of a
fragment approximately 1.5 kb in length. The PCR product was purified using a
QIAquickT"" PCR Purification kit (Qiagen; Valencia, CA) and cloned into the TA
cloning site of pCR2.1-TOPO (Invitrogen; Carlsbad, CA); the ligated product
was
transformed into Max Efficiency E. coli DHSa cells (Life Technologies;
Rockville, MD).
Sequence analysis of the cloned fragment failed to identify a termination
codon for
ytfN. Therefore, oligonucleotide primer KWK-Li-YtfN-12C (SEQ ID N0:21) was
designed and synthesized to be used in a second round of PCR amplification
using
the VectoretteT"" libraries. The above-mentioned conditions for amplification
were
used, and products were visualized by agarose gel electrophoresis. A fragment
approximately 1.6 kb in length was amplified from the Dral library. The PCR
product
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was purified using a QIAquick PCR Purification kit, cloned into pCR2.1-TOPO,
and
subsequently transformed into Max Efficiency E. coli DHSa cells. Sequence
analysis
of the fragment identified a terminal TAA codon indicating the end of the ytfN
gene.
ii) Determination of genomic sequence of complete ytfN gene
Results from the preliminary sequencing described above were used to design
oligonucleotide primers for the specific amplification of two overlapping ytfN
gene
fragments directly from L. infracellularis chromosomal DNA. These products
encompass the entire ytfN gene and were sequenced directly in an attempt to
avoid
introduction of sequence artifacts due to mutations which might arise during
PCR
amplification and subsequent cloning steps. To obtain the first of the two
fragments,
PCR amplifications were carried out in triplicate and contained 100 pMol of
primers
YtfN-D (SEQ. ID N0:45) and YtfN-U (SEQ. ID N0:46), 100 ~g purified chromosomal
DNA, 1x PC2 buffer (Ab Peptides; St. Louis, MO), 200 ~M each dNTP, 15 U
KIenTaq1 (Ab Peptides) and 0.3 U cloned Pfu (Stratagene; La Jolla, CA)
thermostable polymerises in a 100 ~I final sample volume. Conditions for
amplification consisted of denaturation (94°C, 9 min), followed by 40
cycles of
denaturation (94°C, 30 sec), annealing (60°C, 30 sec), and
polymerization (72°C, 4.0
min), and a final extension at 72°C for 7 min. To obtain the second
(overlapping)
fragment, PCR amplifications were carried out in triplicate as described
above,
except that primers KWK-Li-YtfN-12C (SEQ ID N0:21) and KWK-LI-YtfN-15N (SEQ
ID N0:24) were used. Conditions for amplification consisted of denaturation
(94°C,
9 min), followed by 40 cycles of denaturation (94°C, 30 sec), annealing
(55°C, 30
sec), and polymerization (72°C, 2.5 min), and a final extension at
72°C for 7 min.
Following amplification, each set of triplicate samples was pooled and the
specific
product from each was purified (QIAquickT"~ PCR Purification kit). Both
purified DNA
fragments were then subjected to direct sequence analysis using DyeDeoxy
termination reactions on an ABI automated DNA sequencer (Lark Technologies
Inc.,
Houston, TX).
Synthetic oligonucleotide primers (SEQ ID NOs:21, 24, 26-38, 43-46, 51-53, and
55-
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60) were used to sequence both DNA strands of the amplified products.
The ytfN ORF extends from nucleotides 1-4149 of SEQ ID N0:17 and encodes a
1382 amino acid protein (SEQ ID N0:18), having a theoretical molecular weight
of
150,887 Daltons. The sequence of the amino terminus of the encoded protein
resembles a prokaryotic signal sequence (von Heijne, 1985; Nielsen, et al.,
1997),
although the precise site of cleavage is not presently known. The ytfN ORF was
compared against existing nucleotide and protein databases using the Basic
Local
Alignment Search Tool (BLAST) programs (Altschul, et al., 1990). The entry
with
which it shared the greatest homology was a hypothetical 40.5 kDa protein from
Zymomonas mobilis. The second-most-significant homologous sequence identified
was a YtfN homolog from Neisseria meningitidis.
iii) Cloning of recombinant ytfM gene into expression vectors
For the purpose of recombinant protein expression, both the ytfM and ytfN
genes or
fragments thereof were cloned without the sequences encoding their respective
signal peptides.
The ytfM gene was amplified from L. intracellularis chromosomal DNA using
oligonucleotide primers RA202-b (SEQ ID NO: 50) and RA201-b (SEQ ID NO: 49).
For polymerase chain amplification, triplicate 50 ~I reactions were set up
with
eachcontaining 100 ng of chromosomal DNA as template, 1x PC2 buffer, 200 ~.M
each dNTP, 50 pMol each primer, 7.5 U KIenTaq1 and 0.15 U cloned Pfu
thermostable polymerases. Amplification was carried out as follows:
denaturation
(94°C, 9 min); 40 cycles of denaturation (94°C, 30 sec),
annealing (60°C, 30 sec),
and polymerization (72°C, 2.5 min), followed by a final extension at
72°C for 7
minutes. Following amplification, the samples were purified (QIAquickT"" PCR
Purification kit) and pooled. The purified PCR product was cloned directly
into the
TA cloning site of both pBAD-TOPO and pBADlThio-TOPO (Invitrogen). The ligated
products were transformed into Max Efficiency E. coli DHSa cells. The
predicted
amino terminal sequence of the encoded protein expressed from pBAD-TOPO:YtfM
would consist of the vector-encoded sequence MGSGSGDDDDKLALLTM (SEQ ID
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NO: 61 ) followed immediately by the sequence ATSITTS (SEQ ID NO: 62)
beginning
at Alanine-24 of the YtfM ORF (SEQ ID N0:16). A clone containing the
appropriate
plasmid was identified, and purified plasmid was isolated from a small-scale
broth
culture using a QIAprep Spin Miniprep kit (Qiagen). This plasmid was
transformed
into E. coli BL21 (Novagen; Madison, WI) and BL21-CodonPlus-RIL cells
(Stratagene); a clone was identified in each strain that contained the
appropriate
plasmid.
The predicted amino terminal sequence of the encoded fusion protein expressed
from pBAD/Thio-TOPO:YtfM would consist of the thioredoxin protein and a 15
amino
acid residue linker followed immediately by the sequence ATSITTS (SEQ ID NO:
62)
beginning at Alanine-24 of the YtfM ORF (SEQ ID N0:16). A clone containing the
appropriate plasmid was identified, and purified plasmid was isolated from a
small-
scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was
transformed
into E. coli BL21 and BL21-CodonPlus-RIL cells; a clone was identified in each
strain
that contained the appropriate plasmid.
For cloning into pET-30a, the purified PCR product encoding YtfM was digested
with
BamHl and Ncol, then purified using a QIAquickT"" PCR Purification kit. pET-
30a was
also digested with BamHl and Ncol; the linearized plasmid was purified using a
JETsorbT"" kit (Genomed; Frederick, MD) prior to ligation. The ligated product
was
transformed into Max Efficiency E. coli DHSa cells. The predicted amino
terminal
sequence of the encoded fusion protein expressed from pET-30a:YtfM would
consist
of MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAM (SEQ ID
NO: 63) encoded by the vector, followed by the sequence ATSITTS (SEQ ID NO:
62)
beginning at Alanine-24 of the YtfM ORF (SEQ ID N0:16). A clone containing the
appropriate plasmid was identified, and purified plasmid was isolated from a
small-
scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was
transformed
into E, coli BL21(DE3) (Novagen) and BL21-CodonPlus(DE3)-RIL cells
(Stratagene);
a clone was identified in each strain that contained the appropriate plasmid.
The ytfM gene was also amplified from L. intracellularis chromosomal DNA by
PCR
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amplification using oligonucleotide primers RA200 (SEQ ID NO: 47) and RA201
(SEQ ID NO: 48). Duplicate 50 ~.I reactions were set up each containing 100 ng
of
chromosomal DNA as template, 1x PC2 buffer, 200 ~.M each dNTP, 50 pMol each
primer, 7.5 U KIenTaq1 and 0.15 U cloned Pfu thermostable polymerises.
Amplification was carried out as follows: denaturation (94°C, 9 min);
30 cycles of
denaturation (94°C, 30 sec), annealing (60°C, 30 sec), and
polymerization (72°C, 2
min), followed by a final extension at 72°C, for 7 minutes. Following
amplification, the
samples were purified (QIAquickT"" PCR Purification kit) and pooled. The
purified
PCR product was cloned directly into the TA cloning site of pCR2.1-TOPO. The
ligated product was transformed into Max Efficiency E. coli DHSa cells. A
clone
containing the appropriate plasmid was identified, propagated, and plasmid DNA
was
isolated using a QIAprep Spin Miniprep kit. Following digestion of the plasmid
with
EcoRl, a fragment corresponding to by 437 of SEQ ID N0:15 to the EcoRl site in
the
MCS of pCR2.1-TOPO was purified using a JETsorbT"" kit. pET-30a was also
digested with EcoRl, and purified using a QIAquick PCRT"" Purification kit.
The two
fragments were ligated and transformed into Max Efficiency E, coli DHSa cells.
The
predicted amino terminal sequence of the encoded fusion protein would consist
of
MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGS (SEQ
ID NO: 64) encoded by the vector followed by the sequence EFNLSKG (SEQ ID NO:
65) beginning at Aspartate-146 of the YtfM ORF (SEQ ID N0:16). A clone
containing
the plasmid with the gene fragment inserted in the proper orientation was
identified,
and purified plasmid was isolated from a small-scale broth culture using a
QIAprep
Spin Miniprep kit. This plasmid was transformed into E. coli BL21-
CodonPlus(DE3)-
RIL cells; a clone was identified that contained the appropriate plasmid.
iv) Cloning of recombinant ytfN gene into expression vectors
The 5' half of the ytfN gene, excluding that encoding the signal sequence, was
amplified from L. intracellularis chromosomal DNA using oligonucleotide
primers
RA205-b (SEQ ID NO: 53) and RA204-b (SEQ ID NO: 52). For polymerise chain
amplification, triplicate 100 u1 reactions were set up each containing 100 ng
of
chromosomal DNA as template, 1x PC2 buffer, 200 ~.M each dNTP, 100 pMol each
primer, 15 U Klen Taq1 and 0.3 U cloned Pfu thermostable polymerises.
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scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was
transformed
into E. coli BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP cells
(Stratagene); a clone was identified in each strain that contained the
appropriate
plasmid.
Utilizing oligonucleotide primers RA205-b (SEQ ID NO: 53) and KWK-Li-YtfN-
Bglll-3'
(SEQ ID N0:39), an approximate 1 kb fragment encoding the N-terminal portion
of
YtfN, excluding the signal sequence, was amplified by PCR. Triplicate 50 u1
reactions were set up each containing 100 ng of chromosomal DNA as template, 1
x
PC2 buffer, 200 ~.M each dNTP, 50 pMol each primer, 7.5 U KIenTaq1 and 0.15 U
cloned Pfu thermostable polymerases. Amplification was carried out as follows:
denaturation (94°C, 9 min); 40 cycles of denaturation (94°C, 30
sec), annealing
(55°C, 30 sec), and polymerization (72°C, 1 min); foliowed by a
final extension at
72°C for 7 minutes. Following amplification, the samples were purified
(QIAquickT""
PCR Purification kit) and pooled. The purified PCR product was cloned directly
into
the TA cloning site of both pBAD-TOPO and pBAD/Thio-TOPO. The ligated products
were transformed into Max Efficiency E. coli DHSa cells. The predicted' amino
terminal sequence of the protein expressed from pBAD-TOPO would consist of the
vector-encoded sequence MGSGSGDDDDKLALGHM (SEQ ID NO: 66) followed
immediately by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33
of the YtfN ORF (SEQ ID N0:18); the protein would terminate with Isoleucine-
332
of ytfN (SEQ ID N0:18). A clone containing the appropriate plasmid was
identified,
and purified plasmid was isolated from a small-scale broth culture using a
QIAprep
Spin Miniprep kit. This plasmid was transformed into E. coli BL21-CodonPlus-
RIL
cells; a clone was identified that contained the appropriate plasmid.
The predicted amino terminal sequence of the encoded fusion protein expressed
from pBAD/Thio-TOPO:YtfN would consist of the thioredoxin protein and a 15
amino
acid linker followed immediately by the sequence RTSTGIA (SEQ ID NO: 67)
beginning at Arginine-33 of the YtfN ORF (SEQ ID N0:18); again, the
polypeptide
would terminate with Isoleucine-332 of ytfN (SEQ ID N0:18). A clone containing
the
appropriate plasmid was identified, and purified plasmid was isolated from a
small-
Image
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REFERENCES
1. Altuvia, Y., Schueler, O., and Margalit, H. (1995) J. Mol. Biol. 249:244-
250.
2. Amann and Brosius (1985). Gene 40: 183.
3. Anderson, B.J., M.M. Bills, J.R. Egerton, and J.S. Mattick. (1984) Journal
of
Bacteriology 160:748-754.
4. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990, J.
Mol.
Biol. 215:403-410.
5. Ausubel, F. M., Brent, R., Kingston, RE, Moore, D.D., Seidman, J.G., Smith,
J.A., and Struhl, K. (1987). In: Current Protocols in Molecular Biology. Wiley
Interscience (ISBN 047150338).
6. Barker, I.K. and Van Dreumel, A.A. (1985) In OPathology of Domestic
Animals, 3rd Edition, Vol. 2 p. 1-237, eds K.V.F. Jubb, P.C. Kennedy and N.
Palmer. (Academic Press: Orlando).
7. Cole et al. (1985) In: Monoclonal antibodies in cancer therapy, Alan R.
Bliss
Inc., pp 77-96.
8. Dayhof, M.D. (1978) In: Nat. Biomed. Res. Found. Washington D. C. VolS,
Suppl. 3.
9. De Groot, A.S., Carter, E.J., Roberts, C.G.P., Edelson, B.T., Jesdale,
B.M.,
Meister, G.E., Houghten, R.A., Montoya, J., Romulo, R.C., Berzofsky, J.A., and
Ramirezm, B.D.L.L. (1995)Vaccines 96, Cold Spring Harbor Laboratory, Cold
Spring Harbor NY.
10. Devereux, J., Haeberli, P. and Smithies, O. (1984). Nucl. Acids Res. 12:
387-395.
11. Elwell, MR, Chapman, AL and Frenkel, JK (1981 ) Veterinary Pathology 18:
136-139.
12. Fox, JG, Murphy, JC, Otto, G Pecquet-Goad, ME, Larson, QHK and Scott JA
(1989) Veterinary Pathology 26: 515-517.
13. Gabriel, E. Meister, G.E., Caroline, G.P., Roberts, C.G.P., Berzofsky,
J.A., and
De Groot, A.S. (1995) Vaccines 95, Cold Spring Harbor Laboratory, Cold
Spring Harbor NY.
14. Gebhart, C.J., Ward, G.E., Chang, K. And Kurtz, H.J. (1983). American
Journal
of Veterinary Research 44:361-367.
CA 02399276 2002-08-02
_ WO 02/38594 PCT/AU01/01462
81
MISSING AT THE TIME OF PUBLICATION
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-82-
35. Nollau, P., Moser, C. and C. Wagener (1996) BioTechniques 20:784-788.
36. O'Neil, I. P.A. (1970) Veterinary Record 87:742-747.
37. Parker, K.C., Bednarek, M.A., and Coligan, J.E. (1994) J. Immunol.
152:163-175.
38. Portoghese et al. (1990) J. Med. Chem. 33:1714-1720.
39. Reinhartz, A., Alajem, S., Samson, A. and Herzberg, M.(1993). Gene 136:
221-
226.
40. Rowland, A.C. and Lawson, G.H.K. (1976) Veterinary Record 97:178-180.
41. Sambrook, J., E.F. Fritsch, and T. Maniatis. (1989) Molecular cloning. A
laboratory manual. Second edition. Cold Spring Harbour Laboratory, Cold
Spring Harbour, N.Y.
42. Schodeb, TR and Fox JG (1990) Veterinary Pathology 27: 73-80.
43. Shimatake and Rosenberg (1981 ) Nature 292: 128.
44. Stills, H.F. (1991 ). Infection and immunology 59: 3227-3236.
45. Straw, B.E. (1990). Journal ofAmerican Veterinary Medical Association 197:
355-357.
46. Studier and Moffat (1986) J. Mol. Biol. 189: 113.
47. Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994) Nucl. Acids Res.
22:
4673-4680.
48. Vajda, S. and DeLisi, C. (1990) Biopolymers 29:1755-1772.
49. van Regenmortel, M. (1992) Molecular dissection of protein antigens. In:
Structure of antigens, (van Regenmortel M. ed.) CRC Press, London, pp1-27.
50. von Heijne, (1985), J. Mol. Biol. 184: 99-105.
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SEQUENCE LISTING
<110> Australian Pork Limited AND Agriculture Victoria Services Pty Ltd AND
Pfizer Products Inc.
<120> Novel therapeutic compositions for treating infection by Lawsonia spp.
<130>
<150> AU PR1381
<151> 2000-11-10
<150> US 60/249596
<151> 2000-11-17
IS <160> 68
<170> PatentIn Ver. 2.0
<210> 1
<211> 622
<212> DNA
<213> Lawsonia intracellularis
<220>
<221> CDS
<222> (1)..(621)
<400> 1
atg tct gat gat ccc agt aaa aca gag aaa gca acc ccg aaa cga cgt 48
Met Ser Asp Asp Pro Ser Lys Thr Glu Lys Ala Thr Pro Lys Arg Arg
1 5 10 15
cag gaa get cgt tct gaa ggg agt gtc cct aaa tca gaa gag gtt act 96
Gln Glu Ala Arg Ser Glu Gly Ser Val Pro Lys Ser Glu Glu Val Thr
20 25 30
aaa gca ttg act act gca gca ggg atg ctg ggg ctt get att tat tca 144
Lys Ala Leu Thr Thr Ala Ala Gly Met Leu Gly Leu Ala Ile Tyr Ser
35 40 45
ggc gta atg gga cgt cat ttt gaa aca att ttc tac tat att ttt aca 192
Gly Val Met Gly Arg His Phe Glu Thr Ile Phe Tyr Tyr Ile Phe Thr
55 60
45 gaa tca ttt cgg ttt gag gtt aca gca cag tca gta tat get tta ttt 240
Glu Ser Phe Arg Phe Glu Val Thr Ala Gln Ser Val Tyr Ala Leu Phe
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65 70 75 80
att tat gtt get caa gag ata get att tta ttg atg cca ata tta ctt 288
Ile Tyr Val Ala Gln Glu Ile Ala Ile Leu Leu Met Pro Ile Leu Leu
85 90 95
ttt att get gtt acg gca tgg att tca tta cgt gta caa gtt ggt gca 336
Phe Ile Ala Val Thr Ala Trp Ile Ser Leu Arg Val Gln Val Gly Ala
100 105 110
tta tgg act aca aag gtt ttt aaa ttt aaa tgg agt aaa ttt aat ata 384
Leu Trp Thr Thr Lys Val Phe Lys Phe Lys Trp Ser Lys Phe Asn Ile
115 120 125
1$ ata aaa ggg ttg aaa gga atg ttt get tct caa caa aca ctt gtt cga 432
Ile Lys Gly Leu Lys Gly Met Phe Ala Ser Gln Gln Thr Leu Val Arg
130 135 140
ctt tta cgt agt tta gtt caa gta att gtt ata ggt att gtt cca tat 480
Leu Leu Arg Ser Leu Val Gln Val Ile Val Ile Gly Ile Val Pro Tyr
145 150 155 160
atg att ata aaa gga gag ttt tca aac ttt tta cca tta tat tat gca 528
Met Ile Ile Lys Gly Glu Phe Ser Asn Phe Leu Pro Leu Tyr Tyr Ala
165 170 175
agt cct tca ggt gtg gca gat tat atg ctt aat aca gga ata gta ctt 576
Ser Pro Ser Gly Val Ala Asp Tyr Met Leu Asn Thr Gly Ile Val Leu
180 185 190
gtt tta tat acg cta att cct atg aca att att gca gtc gca gat c 622
Val Leu Tyr Thr Leu Ile Pro Met Thr Ile Ile Ala Val Ala Asp
195 200 205
<210> z
<211> zo7
<212> PRT
<213> Lawsonia intracellularis
<400> 2
Met Ser Asp Asp Pro Ser Lys Thr Glu Lys Ala Thr Pro Lys Arg Arg
1 5 10 15
Gln Glu Ala Arg Ser Glu Gly Ser Val Pro Lys Ser Glu Glu Val Thr
20 25 30
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Lys Ala Leu Thr Thr Ala Ala Gly Met Leu Gly Leu Ala Ile Tyr Ser
35 40 45
Gly Val Met Gly Arg His Phe Glu Thr Ile Phe Tyr Tyr Ile Phe Thr
50 55 60
Glu Ser Phe Arg Phe Glu Val Thr Ala Gln Ser Val Tyr Ala Leu Phe
65 70 75 80
Ile Tyr Val Ala Gln Glu Ile Ala Ile Leu Leu Met Pro Ile Leu Leu
85 90 95
Phe Ile Ala Val Thr Ala Trp Ile Ser Leu Arg Val Gln Val Gly Ala
loo los llo
Leu Trp Thr Thr Lys Val Phe Lys Phe Lys Trp Ser Lys Phe Asn Ile
115 120 125
Ile Lys Gly Leu Lys Gly Met Phe Ala Ser Gln Gln Thr Leu Val Arg
130 135 140
Leu Leu Arg Ser Leu Val Gln Val Ile Val Ile Gly Ile Val Pro Tyr
145 150 155 160
Met Ile Ile Lys Gly Glu Phe Ser Asn Phe Leu Pro Leu Tyr Tyr Ala
165 170 175
Ser Pro Ser Gly Val Ala Asp Tyr Met Leu Asn Thr Gly Ile Val Leu
leo 18s 190
Val Leu Tyr Thr Leu Ile Pro Met Thr Ile Ile Ala Val Ala Asp
195 200 205
<210> 3
<211> 789
<212> DNA
<213> Lawsonia intracellularis
<zzo>
<221> CDS
<222> (1)..(786)
<400> 3
atg aat tta ttt aat ttt gat cct agt atg ttt ctt agt ttt tta ctt 48
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Met Asn Leu Phe Asn Phe Asp Pro Ser Met Phe Leu Ser Phe Leu Leu
1 5 10 15
aca ttt tta cgt att agt gtt gtc tta ttt atg ctt cct ttt ttt tct 96
Thr Phe Leu Arg Ile Ser Val Val Leu Phe Met Leu Pro Phe Phe Ser
20 25 30
att gat ggt ttt cct aat atg tta aaa gca tca ata get ctt att cta 144
Ile Asp Gly Phe Pro Asn Met Leu Lys Ala Ser Ile Ala Leu Ile Leu
1~ 35 40 45
act ata gtt ctt tgg ggg cgt ctt tct ctt tca gga aca caa atg cca 192
Thr Ile Val Leu Trp Gly Arg Leu Ser Leu Ser Gly Thr Gln Met Pro
50 55 60
IS
gcg cat cct ttc gat cta gta ttg tta atc ata agc gag gtt ttt ctt 240
Ala His Pro Phe Asp Leu Val Leu Leu Ile Ile Ser Glu Val Phe Leu
65 70 75 80
ggt att gta ttg ggg ctt gcg gta aac ttt ttc ttt gca gga att caa 288
Gly Ile Val Leu Gly Leu Ala Val Asn Phe Phe Phe Ala Gly Ile Gln
85 90 95
get ggg gga gaa att ctt get aca caa atg ggg ttt aca atg att acg 336
25 Ala Gly Gly Glu Ile Leu Ala Thr Gln Met Gly Phe Thr Met Ile Thr
100 105 110
ctt gca gac cca tta act ggt aac acc aca ggt ttt att gca cat ttt 384
Leu Ala Asp Pro Leu Thr Gly Asn Thr Thr Gly Phe Ile Ala His Phe
3~ 115 120 125
ctt tat atg gtt get aca tta gtt ttt ctt get ctt aat ggc cat ttg 432
Leu Tyr Met Val Ala Thr Leu Val Phe Leu Ala Leu Asn Gly His Leu
130 135 140
ttt ctt ata aaa get ttt aca tat act ttt aaa atg gtt cca gca gga 480
Phe Leu Ile Lys Ala Phe Thr Tyr Thr Phe Lys Met Val Pro Ala Gly
145 150 155 160
gga ctt gtt gta aga gaa att tta ttg agt gaa ctt ctt aat atg gca 528
Gly Leu Val Val Arg Glu Ile Leu Leu Ser Glu Leu Leu Asn Met Ala
165 170 175
ggg atg att ttt gtt ttt gcc tta cat gtt gcg gca cca gtt atg tca 576
Gly Met Ile Phe Val Phe Ala Leu His Val Ala Ala Pro Val Met Ser
180 185 190
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get ctt ttt tta gta gag atc tct tta gga ctt atg gca aga get get 624
Ala Leu Phe Leu Val Glu Ile Ser Leu Gly Leu Met Ala Arg Ala Ala
195 200 205
cct cag att cat att atg gaa gtt gga ttt cct gta aaa att ggt gta 672
Pro Gln Ile His Ile Met Glu Val Gly Phe Pro Val Lys Ile Gly Val
210 215 220
gga ttt ttt ttc att gga cta tta ttt act atc tta tca aaa gaa acc 720
Gly Phe Phe Phe Ile Gly Leu Leu Phe Thr Ile Leu Ser Lys Glu Thr
225 230 235 240
tat cga ttt att gca ggc cta gag gga cta ttt ttt aac tta ctt act 768
IS Tyr Arg Phe Ile Ala Gly Leu Glu Gly Leu Phe Phe Asn Leu Leu Thr
245 250 255
gta atg ggt agt gga aaa tag 789
Val Met Gly Ser Gly Lys
260
<210> 4
<211> 262
<212> PRT
<213> Lawsonia intracellularis
<400> 4
Met Asn Leu Phe Asn Phe Asp Pro Ser Met Phe Leu Ser Phe Leu Leu
1 5 10 15
Thr Phe Leu Arg Ile Ser Val Val Leu Phe Met Leu Pro Phe Phe Ser
20 25 30
Ile Asp Gly Phe Pro Asn Met Leu Lys Ala Ser Ile Ala Leu Ile Leu
35 40 45
Thr Ile Val Leu Trp Gly Arg Leu Ser Leu Ser Gly Thr Gln Met Pro
50 55 60
Ala His Pro Phe Asp Leu Val Leu Leu Ile Ile Ser Glu Val Phe Leu
65 70 75 80
Gly Ile Val Leu Gly Leu Ala Val Asn Phe Phe Phe Ala Gly Ile Gln
85 90 95
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Ala Gly Gly Glu Ile Leu Ala Thr Gln Met Gly Phe Thr Met Ile Thr
100 105 110
Leu Ala Asp Pro Leu Thr Gly Asn Thr Thr Gly Phe Ile Ala His Phe
115 lzo lzs
Leu Tyr Met Val Ala Thr Leu Val Phe Leu Ala Leu Asn Gly His Leu
130 135 140
Phe Leu Ile Lys Ala Phe Thr Tyr Thr Phe Lys Met Val Pro Ala Gly
145 150 155 160
Gly Leu Val Val Arg Glu Ile Leu Leu Ser Glu Leu Leu Asn Met Ala
165 170 175
1$
Gly Met Ile. Phe Val Phe Ala Leu His Val Ala Ala Pro Val Met Ser
180 185 190
Ala Leu Phe Leu Val Glu Ile Ser Leu Gly Leu Met Ala Arg Ala Ala
195 200 205
Pro Gln Ile His Ile Met Glu Val Gly Phe Pro Val Lys Ile Gly Val
210 215 220
2$ Gly Phe Phe Phe Ile Gly Leu Leu Phe Thr Ile Leu Ser Lys Glu Thr
225 230 235 240
Tyr Arg Phe Ile Ala Gly Leu Glu Gly Leu Phe Phe Asn Leu Leu Thr
245 250 255
Val Met Gly Ser Gly Lys
260
3$ <210> s
<211> 1371
<212> DNA
<213> Lawsonia intracellularis
<220>
<221> CDS
<222> (1)..(1368)
<400> 5
4$ atg tca gca cgt ata ctt att ata gat gat gaa gac tct att aga ttt 48
Met Ser Ala Arg Ile Leu Ile Ile Asp Asp Glu Asp Ser Ile Arg Phe
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1 5 10 15
ca ttg aaa gga att ttt gaa gat gag ggc cat gaa gtt tta gaa aga 96
Ser Leu Lys Gly Ile Phe Glu Asp Glu Gly His Glu Val Leu Glu Arg
20 25 30
get tca gca gaa gaa gga ctt aag tgt gtt gat gta gag tct cca gat 144
Ala Ser Ala Glu Glu Gly Leu Lys Cys Val Asp Val Glu Ser Pro Asp
35 40 45
ctt gtt ttt ctt gat att tgg ctt cct ggg atg gat ggt ctt atg get 192
Leu Val Phe Leu Asp Ile Trp Leu Pro Gly Met Asp Gly Leu Met Ala
50 55 60
IS tta gac cat att cag get ctt cat cag gaa tta cct gtt att atg att 240
Leu Asp His Ile Gln Ala Leu His Gln Glu Leu Pro Val Ile Met Ile
65 70 75 80
tca ggt cat gcc aca att gaa act get gta aca get atc cgt caa ggt 288
Ser Gly His Ala Thr Ile Glu Thr Ala Val Thr Ala Ile Arg Gln Gly
85 90 95
get tat gat ttt att gaa aag cct ctt tct ttg gaa aaa gtc ctt att 336
Ala Tyr Asp Phe Ile Glu Lys Pro Leu Ser Leu Glu Lys Val Leu Ile
loo l05 llo
aca get aat aga get ata gaa aca gta aga tta aga agg gaa aac aaa 384
Thr Ala Asn Arg Ala Ile Glu Thr Val Arg Leu Arg Arg Giu Asn Lys
115 120 125
tta cta cgt act gta tta cct gag gag agt gag ttt ata gga cag tct 432
Leu Leu Arg Thr Val Leu Pro Glu Glu Ser Glu Phe Ile Gly Gln Ser
130 135 140
cct gtt atc tta aaa ttt aaa agt tta tta tca cag gtc get cca aca 480
Pro Val Ile Leu Lys Phe Lys Ser Leu Leu Ser Gln Val Ala Pro Thr
145 150 155 160
gat get tgg gta cta ctt aca gga gag aat ggt aca ggt aaa gag tta 528
Asp Ala Trp Val Leu Leu Thr Gly Glu Asn Gly Thr Gly Lys Glu Leu
165 170 175
get gca caa gca ttg cac aaa gga agc tca cga tat caa aaa cca ttt 576
Ala Ala Gln Ala Leu His Lys Gly Ser Ser Arg Tyr Gln Lys Pro Phe
180 1e5 190
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_g_
ata get gtt aat tgt get get atc cct gaa gaa ttg att gaa agc gaa 624
Ile Ala Val Asn Cys Ala Ala Ile Pro Glu Glu Leu Ile Glu Ser Glu
195 200 205
$ cta ttt ggt cat gaa aaa ggg gcc ttt act ggt gcc gat get tct cgt 672
Leu Phe Gly His Glu Lys Gly Ala Phe Thr Gly Ala Asp Ala Ser Arg
210 215 220
gca ggt cgt ttt gag ttg gca cat aaa gga aca tta ttt ctt gat gaa 720
Ala Gly Arg Phe Glu Leu Ala His Lys Gly Thr Leu Phe Leu Asp Glu
225 230 235 240
ata gga gat atg agt tta aaa aca caa gca aaa att ttg cgt att ttg 768
Ile Gly Asp Met Ser Leu Lys Thr Gln Ala Lys Ile Leu Arg Ile Leu
1$ 245 250 255
caa gaa caa tgt ttt gaa aaa att ggt agt gtt aga act att aaa gtt 816
Gln Glu Gln Cys Phe Glu Lys Ile Gly Ser Val Arg Thr Ile Lys Val
260 265 270
gat gta aga gtt att gca gca aca aat aag aat ctt gaa gac get att 864
Asp Val Arg Val Ile Ala Ala Thr Asn Lys Asn Leu Glu Asp Ala Ile
275 280 285
2$ agc gat gga aca ttt cgt caa gat ttg tat tat cgc tta cga gtt gtt 912
Ser Asp Gly Thr Phe Arg Gln Asp Leu Tyr Tyr Arg Leu Arg Val Val
290 295 300
cca ttg cat ctt ccc cct ctt cgt gaa cgt gat tct gat att gag cta 960
Pro Leu His Leu Pro Pro Leu Arg Glu Arg Asp Ser Asp Ile Glu Leu
305 310 315 320
tta tta aat agg ttt gtg att cag ttg agt aaa cgt tat aga cgt gag 1008
Leu Leu Asn Arg Phe Val Ile Gln Leu Ser Lys Arg Tyr Arg Arg Glu
3$ 325 330 335
ccg cct att ttt tta gat gag gtc ttc cct gta ttg aaa caa tat tgt 1056
Pro Pro Ile Phe Leu Asp Glu Val Phe Pro Val Leu Lys Gln Tyr Cys
340 345 350
tgg cca ggg aat gta aga gaa tta ctt aat ttt gta gaa cga atg gtt 1104
Trp Pro Gly Asn Val Arg Glu Leu Leu Asn Phe Val Glu Arg Met Val
355 360 365
4$ att ctt tat tca ggg aag aaa gta tgt ttg aca gat cct aag gta aaa 1152
Ile Leu Tyr Ser Gly Lys Lys Val Cys Leu Thr Asp Pro Lys Val Lys
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370 375 380
agc aat tta aaa tat tta ccc aag aaa ttt tct tcc cat tat aac ttt 1200
Ser Asn Leu Lys Tyr Leu Pro Lys Lys Phe Ser Ser His Tyr Asn Phe
385 390 395 400
ctt ccc gat ata gat ttt aac cag get aaa ata get ttt gaa cca aaa 1248
Leu Pro Asp Ile Asp Phe Asn Gln Ala Lys Ile Ala Phe Glu Pro Lys
405 410 415
1
ttt tta act gaa aaa tta cat get tat caa gga aat att acc cga tta 1296
Phe Leu Thr Glu Lys Leu His Ala Tyr Gln Gly Asn Ile Thr Arg Leu
420 425 430
1$ gca gaa get att gga ctt gaa aga agt tat tta tat aga aag cta aaa 1344
Ala Glu Ala Ile Gly Leu Glu Arg Ser Tyr Leu Tyr Arg Lys Leu Lys
435 440 445
agc tat ggt att tat ctg tct gag tga 1371
Ser Tyr Gly Ile Tyr Leu Ser Glu
450 455
<210> 6
25 <211> 456
<212> PRT
<213> Lawsonia intracellularis
<400> 6
3~ Met Ser Ala Arg Ile Leu Ile Ile Asp Asp Glu Asp Ser Ile Arg Phe
1 5 10 15
Ser Leu Lys Gly Ile Phe Glu Asp Glu Gly His Glu Val Leu Glu Arg
20 25 30
Ala Ser Ala Glu Glu Gly Leu Lys Cys Val Asp Val Glu Ser Pro Asp
35 40 45
Leu Val Phe Leu Asp Ile Trp Leu Pro Gly Met Asp Gly Leu Met Ala
50 55 60
Leu Asp His Ile Gln Ala Leu His Gln Glu Leu Pro Val Ile Met Ile
65 70 75 80
Ser Gly His Ala Thr Ile Glu Thr Ala Val Thr Ala Ile Arg Gln Gly
85 90 95
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Ala TyrAspPheIleGluLysProLeuSerLeu GluLysValLeuIle
100 105 110
Thr AlaAsnArgAlaIleGluThrValArgLeu ArgArgGluAsnLys
115 120 125
Leu LeuArgThrValLeuProGluGluSerGlu PheIleGlyGlnSer
130 135 140
Pro ValIleLeuLysPheLysSerLeuLeuSer GlnValAlaProThr
145 150 155 160
Asp AlaTrpValLeuLeuThrGlyGluAsnGly ThrGlyLysGluLeu
165 170 175
Ala AlaGlnAlaLeuHisLysGlySerSerArg TyrGlnLysProPhe
180 185 190
Ile AlaValAsnCysAlaAlaIleProGluGlu LeuIleGluSerGlu
195 200 205
Leu PheGlyHisGluLysGlyAlaPheThrGly AlaAspAlaSerArg
210 215 220
Ala GlyArgPheGluLeuAlaHisLysGlyThr LeuPheLeuAspGlu
225 230 235 240
Ile GlyAspMetSerLeuLysThrGlnAlaLys IleLeuArgIleLeu
245 250 255
Gln GluGlnCysPheGluLysIleGlySerVal ArgThrIleLysVal
260 265 270
Asp ValArgValIleAlaAlaThrAsnLysAsn LeuGluAspAlaIle
275 280 285
Ser AspGlyThrPheArgGlnAspLeuTyrTyr ArgLeuArgValVal
290 295 300
Pro LeuHisLeuProProLeuArgGluArgAsp SerAspIleGluLeu
305 310 315 320
Leu LeuAsnArgPheValIleGlnLeuSerLys ArgTyrArgArgGlu
325 330 335
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Pro Pro Ile Phe Leu Asp Glu Val Phe Pro Val Leu Lys Gln Tyr Cys
340 345 350
Trp Pro Gly Asn Val Arg Glu Leu Leu Asn Phe Val Glu Arg Met Val
355 360 365
Ile Leu Tyr Ser Gly Lys Lys Val Cys Leu Thr Asp Pro Lys Val Lys
370 375 380
l~ Ser Asn Leu Lys Tyr Leu Pro Lys Lys Phe Ser Ser His Tyr Asn Phe
385 390 395 400
Leu Pro Asp Ile Asp Phe Asn Gln Ala Lys Ile Ala Phe Glu Pro Lys
405 410 415
Phe Leu Thr Glu Lys Leu His Ala Tyr Gln Gly Asn Ile Thr Arg Leu
420 425 430
Ala Glu Ala Ile Gly Leu Glu Arg Ser Tyr Leu Tyr Arg Lys Leu Lys
2~ 435 440 445
Ser Tyr Gly Ile Tyr Leu Ser Glu
450 455
<210> 7
<211> 412
<212> DNA
<213> Lawsonia intracellularis
<220>
<221> CDS
<222> (1)..(411)
<400> 7
aaa caa att gat ata atc att agt ggg get acg ata act ctt gaa cgt 48
Lys Gln Ile Asp Ile Ile Ile Ser Gly Ala Thr Ile Thr Leu Glu Arg
1 5 10 15
aat ctt caa gtc aat ttt tct aac cca tac cat caa aca gat att gaa 96
Asn Leu Gln Val Asn Phe Ser Asn Pro Tyr His Gln Thr Asp Ile Glu
20 25 30
gtc ctg get aat gca aaa aaa gtt aaa ggg atg aag ttt cca caa gac 144
Val Leu Ala Asn Ala Lys Lys Val Lys Gly Met Lys Phe Pro Gln Asp
35 40 45
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ttt aat aaa cct gaa gtt ata gtt get ata cgt aat ggt agt aca gtt 192
Phe Asn Lys Pro Glu Val Ile Val Ala Ile Arg Asn Gly Ser Thr Val
50 55 60
att act cct gca aag caa ctt ctt cct aaa gca tct ttt aga ctc ttt 240
Ile Thr Pro Ala Lys Gln Leu Leu Pro Lys Ala Ser Phe Arg Leu Phe
65 70 75 80
gat gat gaa gtt gca tct ata aaa gat gta gaa tct gga caa tca cat 288
Asp Asp Glu Val Ala Ser Ile Lys Asp Val Glu Ser Gly Gln Ser His
85 90 95
ata tta tta get tca gca cca tta cca gcg att caa get ata aac tca 336
Ile Leu Leu Ala Ser Ala Pro Leu Pro Ala Ile Gln Ala Ile Asn Ser
loo l05 llo
aat ggc aac ctt att cgt tta gat aca ctc ccc att act cat caa tct 384
Asn Gly Asn Leu Ile Arg Leu Asp Thr Leu Pro Ile Thr His Glr_ Ser
115 120 125
gta gga ttt gca ata aag aag gga gat c 412
Val Gly Phe Ala Ile Lys Lys Gly Asp
130 135
<210> s
<211> 137
<212> PRT
<213> Lawsonia intracellularis
<400> 8
Lys Gln Ile Asp Ile Ile Ile Val Gly Ala Thr Ile Thr Leu Glu Arg
1 5 10 15
Asn Leu Gln Val Asn Phe Ser Asn Pro Tyr His Gln Thr Asp Ile Glu
20 25 30
Val Leu Ala Asn Ala Lys Lys Val Lys Gly Met Lys Phe Pro Gln Asp
35 40 45
Phe Asn Lys Pro Glu Val Ile Val Ala Ile Arg Asn Gly Ser Thr Val
55 60
Ile Thr Pro Ala Lys Gln Leu Leu Pro Lys Ala Ser Phe Arg Leu Phe
45 65 70 75 80
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Asp Asp Glu Val Ala Ser Ile Lys Asp Val Glu Ser Gly Gln Ser His
85 90 95
Ile Leu Leu Ala Ser Ala Pro Leu Pro Ala Ile Gln Ala Ile Asn Ser
100 105 110
Asn Gly Asn Leu Ile Arg Leu Asp Thr Leu Pro Ile Thr His Gln Ser
115 120 125
1~ Val Gly Phe Ala Ile Lys Lys Gly Asp
130 135
<210> 9
15 <211> 849
<212> DNA
<213> Lawsonia intracellularis
<220>
20 <221> CDS
<222> (1)..(846)
<400> 9
atg tat att att att gga tac ttt att gtt att get tcc att att gga 48
25 Met Tyr Ile Ile Ile Gly Tyr Phe Ile Val Ile Ala Ser Ile Ile Gly
1 5 10 15
ggc tac ctt atg get aaa ggg aat ctt get tta ctc ttt caa cct gca 96
Gly Tyr Leu Met Ala Lys Gly Asn Leu Ala Leu Leu Phe Gln Pro Ala
3~ 20 25 30
gaa ctt gtt atc att att ggg gca gca tta ggt get ttt ttt get tca 144
Glu Leu Val Ile Ile Ile Gly Ala Ala Leu Gly Ala Phe Phe Ala Ser
35 40 45
cag acg aaa tat tca ttt act ctg gtc att aaa aat tta tca cac att 192
Gln Thr Lys Tyr Ser Phe Thr Leu Val Ile Lys Asn Leu Ser His Ile
50 55 60
ttt ggc gat cca aac agt aca aaa ata aaa tac ctt gaa aca ctt gcc 240
Phe Gly Asp Pro Asn Ser Thr Lys Ile Lys Tyr Leu Glu Thr Leu Ala
65 70 75 80
ctt ctc tat gga ctt ttc tta aaa atg aat aga gaa ggt gtc att agt 288
Leu Leu Tyr Gly Leu Phe Leu Lys Met Asn Arg Glu Gly Val Ile Ser
85 90 95
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ata gaa agt gat ata gaa aaa cct gaa tca agt cct atc ttt agt aaa 336
Ile Glu Ser Asp Ile Glu Lys Pro Glu Ser Ser Pro Ile Phe Ser Lys
100 105 110
tac cct aca att gta aaa gat act aaa gtt gtt gcc ttt att gca gat 384
Tyr Pro Thr Ile Val Lys Asp Thr Lys Val Val Ala Phe Ile Ala Asp
115 120 125
aca tta cga gtt tat ctg aca aca ggt gca cca gaa gat ata gat aac 432
Thr Leu Arg Val Tyr Leu Thr Thr Gly Ala Pro Glu Asp Ile Asp Asn
130 135 140
ctc atg gaa tct gac atg aaa att aca cac gaa gaa gaa tta tta cct 480
Leu Met Glu Ser Asp Met Lys Ile Thr His Glu Glu Glu Leu Leu Pro
145 150 155 160
gca cat tcc atc agc cat atg gca gag tcg cta cca gga atg ggt att 528
Ala His Ser Ile Ser His Met Ala Glu Ser Leu Pro Gly Met Gly Ile
165 170 175
gtt get gca gta tta ggt gtt gtt att acc atg gga aaa att aat gag 576
Val Ala Ala Val Leu Gly Val Val Ile Thr Met Gly Lys Ile Asn Glu
180 185 190
cct cca gaa gtc ctt ggg cat tat att gga gca get ttg gtt ggt aca 624
Pro Pro Glu Val Leu Gly His Tyr Ile Gly Ala Ala Leu Val Gly Thr
195 200 205
ttt ata ggt att ctt ttc tgt tat ggt ttt ttt gga cct atg ggt tca 672
Phe Ile Gly Ile Leu Phe Cys Tyr Gly Phe Phe Gly Pro Met Gly Ser
210 215 220
aag ctt gaa acc tct gca gaa gaa gca cat ttt tat tat aat tcc att 720
Lys Leu Glu Thr Ser Ala Glu Glu Ala His Phe Tyr Tyr Asn Ser Ile
225 230 235 240
aaa gaa get gtt gca get get atc cga ggt tct aca cca atg ata gca 768
Lys Glu Ala Val Ala Ala Ala Ile Arg Gly Ser Thr Pro Met Ile Ala
4~ 245 250 255
gta gaa tat gga aga cgt gcc ata cct aat aca ttt cgt cca tca ttt 816
Val Glu Tyr Gly Arg Arg Ala Ile Pro Asn Thr Phe Arg Pro Ser Phe
260 265 270
tcg gaa atg gaa gaa cgt cta aaa aca gga taa 849
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-IS-
Ser Glu Met Glu Glu Arg Leu Lys Thr Gly
275 280
<210> 10
<211> 282
<212> PRT
<213> Lawsonia intracellularis
<400> 10
Met Tyr Ile Ile Ile Gly Tyr Phe Ile Val Ile Ala Ser Ile Ile Gly
1 5 10 15
Gly Tyr Leu Met Ala Lys Gly Asn Leu Ala Leu Leu Phe Gln Pro Ala
IS 20 25 30
Glu Leu Val Ile Ile Ile Gly Ala Ala Leu Gly Ala Phe Phe Ala Ser
35 40 45
Gln Thr Lys Tyr Ser Phe Thr Leu Val Ile Lys Asn Leu Ser His Ile
50 55 60
Phe Gly Asp Pro Asn Ser Thr Lys Ile Lys Tyr Leu Glu Thr Leu Ala
65 70 75 80
Leu Leu Tyr Gly Leu Phe Leu Lys Met Asn Arg Glu Gly Val Ile Ser
85 90 95
Ile Glu Ser Asp Ile Glu Lys Pro Glu Ser Ser Pro Ile Phe Ser Lys
loo l05 llo
Tyr Pro Thr Ile Val Lys Asp Thr Lys Val Val Ala Phe Ile Ala Asp
115 120 125
Thr Leu Arg Val Tyr Leu Thr Thr Gly Ala Pro Glu Asp Ile Asp Asn
130 135 140
Leu Met Glu Ser Asp Met Lys Ile Thr His Glu Glu Glu Leu Leu Pro
145 150 155 160
Ala His Ser Ile Ser His Met Ala Glu Ser Leu Pro Gly Met Gly Ile
165 170 175
Val Ala Ala Val Leu Gly Val Val Ile Thr Met Gly Lys Ile Asn Glu
180 185 190
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Pro Pro Glu Val Leu Gly His Tyr Ile Gly Ala Ala Leu Val Gly Thr
195 200 205
Phe Ile Gly Ile Leu Phe Cys Tyr Gly Phe Phe Gly Pro Met Gly Ser
210 215 220
Lys Leu Glu Thr Ser Ala Glu Glu Ala His Phe Tyr Tyr Asn Ser Ile
225 230 235 240
Lys Glu Ala Val Ala Ala Ala Ile Arg Gly Ser Thr Pro Met Ile Ala
245 250 255
Val Glu Tyr Gly Arg Arg Ala Ile Pro Asn Thr Phe Arg Pro Ser Phe
260 265 270
Ser Glu Met Glu Glu Arg Leu Lys Thr Gly
275 280
<210> 11
<211> 717
<212> DNA
<213> Lawsonia intracellularis
<220>
<221> CDS
<222> (1)..(714)
<400> 11
atg tct ggc tca tgg aaa gtg get tat gca gac ttt gtt aca get atg 48
Met Ser Gly Ser Trp Lys Val Ala Tyr Ala Asp Phe Val Thr Ala Met
1 5 10 15
atg get ttc ttt cta ctg atg tgg att ctt gca atg aca ccc cct gag 96
Met Ala Phe Phe Leu Leu Met Trp Ile Leu Ala Met Thr Pro Pro Glu
20 25 30
gtt aaa gaa ggt ctt get gca tat ttt tct tca tct gat get aca ttt 144
Val Lys Glu Gly Leu Ala Ala Tyr Phe Ser Ser Ser Asp Ala Thr Phe
35 40 45
aaa aca cct gat agt tcg cca atc tct aac aat cct ctt atc aac caa 192
Lys Thr Pro Asp Ser Ser Pro Ile Ser Asn Asn Pro Leu Ile Asn Gln
55 60
ata gat aaa ctt gat act cga caa tta aaa att aat gaa aca gaa caa 240
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Ile Asp Lys Leu Asp Thr Arg Gln Leu Lys Ile Asn Glu Thr Glu Gln
65 70 75 80
tct cat tat get ctt get aat aaa tta aaa aaa atg tta atg get gat 288
Ser His Tyr Ala Leu Ala Asn Lys Leu Lys Lys Met Leu Met Ala Asp
85 90 95
get atc cca cag tca gca aca gga ata agt get gac gat gtt ggt gta 336
Ala Ile Pro Gln Ser Ala Thr Gly Ile Ser Ala Asp Asp Val Gly Val
1~ 100 105 110
tta tta cgt gta aat tct aat tcc acg ttt ttt cct ggt aca gca act 384
Leu Leu Arg Val Asn Ser Asn Ser Thr Phe Phe Pro Gly Thr Ala Thr
115 120 125
ctt aca ccc gaa ggg aaa aaa gtt atg gga act gtt tta gcc gtt ctc 432
Leu Thr Pro Glu Gly Lys Lys Val Met Gly Thr Val Leu Ala Val Leu
130 135 140
cgt gaa tat aat ctt tac ctt gtg ata cgt ggc cat get gat att ggt 480
Arg Glu Tyr Asn Leu Tyr Leu Val Ile Arg Gly His Ala Asp Ile Gly
145 150 155 160
gaa ata aca aaa ggc agc cct ttt get tct aac tgg gaa ctt tca gga 528
Glu Ile Thr Lys Gly Ser Pro Phe Ala Ser Asn Trp Glu Leu Ser Gly
165 170 175
get cgt gca get gca get gca cag tat ctt gta gag cac ggg ata aag 576
Ala Arg Ala Ala Ala Ala Ala Gln Tyr Leu Val Glu His Gly Ile Lys
3~ 180 185 190
get tca cga att cgc tct gta gga tat gca gat aca aga cct cta gaa 624
Ala Ser Arg Ile Arg Ser Val Gly Tyr Ala Asp Thr Arg Pro Leu Glu
195 200 205
cct agt tct cct gaa gga agt aca aaa aat cgt cgt ata gaa ttc tat 672
Pro Ser Ser Pro Glu Gly Ser Thr Lys Asn Arg Arg Ile Glu Phe Tyr
210 215 220
4~ ttt cat cgg cca gaa gtt atg tct tat ggc gtt gta tat taa tag 717
Phe His Arg Pro Glu Val Met Ser Tyr Gly Val Val Tyr
225 230 235
<210> 1z
<211> 237
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<212>
PRT
<213> wsonia ntracell ularis
La i
<400>
12
Met SerGlySerTrpLysValAlaTyrAlaAspPheValThrAlaMet
1 5 10 15
Met AlaPhePheLeuLeuMetTrpIleLeuAlaMetThrProProGlu
20 25 30
Val LysGluGlyLeuAlaAlaTyrPheSerSerSerAspAlaThrPhe
35 40 45
Lys ThrProAspSerSerProIleSerAsnAsnProLeuIleAsnGln
IS 50 55 60
Ile AspLysLeuAspThrArgGlnLeuLysIleAsnGluThrGluGln
65 70 75 80
Ser HisTyrAlaLeuAlaAsnLysLeuLysLysMetLeuMetAlaAsp
85 90 95
Ala IleProGlnSerAlaThrGlyIleSerAlaAspAspValGlyVal
100 105 110
Leu LeuArgValAsnSerAsnSerThrPhePheProGlyThrAlaThr
115 120 125
Leu ThrProGluGlyLysLysValMetGlyThrValLeuAlaValLeu
130 135 140
Arg GluTyrAsnLeuTyrLeuValIleArgGlyHisAlaAspIleGly
145 150 155 160
Glu IleThrLysGlySerProPheAla-SerAsnTrpGluLeuSerGly
165 170 175
Ala ArgAlaAlaAlaAlaAlaGlnTyrLeuValGluHisGlyIleLys
180 185 190
Ala SerArgIleArgSerValGlyTyrAlaAspThrArgProLeuGlu
195 200 205
Pro SerSerProGluGlySerThrLysAsnArgArgIleGluPheTyr
21o zls 2zo
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Phe His Arg Pro Glu Val Met Ser Tyr Gly Val Val Tyr
225 230 235
<210> 13
<211> 1047
<212> DNA
<213> Lawsonia intracellularis
<2zo>
<221> CDS
<222> (1)..(1044)
<400> 13
1$ atg ata atc ctt tta gga act gtt ttt ctt att gtt ctt atc tct gca 48
Met Ile Ile Leu Leu Gly Thr Val Phe Leu Ile Val Leu Ile Ser Ala
1 5 10 15
tta tgc tca atg atg gaa get get ata tac tct atc cct att act tat 96
Leu Cys Ser Met Met Glu Ala Ala Ile Tyr Ser Ile Pro Ile Thr Tyr
20 25 30
att gaa cac ctt cgt gaa cag gga agc aaa aaa gga gaa aaa ctt tat 144
Ile Glu His Leu Arg Glu Gln Gly Ser Lys Lys Gly Glu Lys Leu Tyr
35 40 45
tat tta cat agt aat att gat cag cct att aca gcc gta tta ata ttg 192
Tyr Leu His Ser Asn tle Asp Gln Pro Ile Thr Ala Val Leu Ile Leu
50 55 60
aat act ata gca aat act get gga get gcc ctt get gga gca att get 240
Asn Thr Ile Ala Asn Thr Ala Gly Ala Ala Leu Ala Gly Ala Ile Ala
65 70 75 80
aca aca aca ctt cat gaa tct act aag cct ttc ttt gca gca atc ctc 288
Thr Thr Thr Leu His Glu Ser Thr Lys Pro Phe Phe Ala Ala Ile Leu
85 90 95
acc ttg ctt att tta get ttt ggg gaa att ata cct aaa aca cta ggt 336
Thr Leu Leu Ile Leu Ala Phe Gly Glu Ile Ile Pro Lys Thr Leu Gly
100 105 110
gtt get tac tct aaa cgt att get ata att ctc ctt aat cct ctc tct 384
Val Ala Tyr Ser Lys Arg Ile Ala Ile Ile Leu Leu Asn Pro Leu Ser
115 120 125
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att ctt ata gtt act tta aaa ccc ctt att atg ctt tca agc tac tta 432
Ile Leu Ile Val Thr Leu Lys Pro Leu Ile Met Leu Ser Ser Tyr Leu
130 135 140
$ aca cga ctt gtt tca cct cga aaa cgt cct aca gtt aca gaa gat gac 480
Thr Arg Leu Val Ser Pro Arg Lys Arg Pro Thr Val Thr Glu Asp Asp
145 150 155 160
atc cgt gca ctt aca agt ctt tcc aga gag tct ggt cgt att aag cca 528
Ile Arg Ala Leu Thr Ser Leu Ser Arg Glu Ser Gly Arg Ile Lys Pro
165 170 175
tat gaa gaa cat gtc ata aaa aat atc ctt agt ctt gat tta aaa tat 576
Tyr Glu Glu His Val Ile Lys Asn Ile Leu Ser Leu Asp Leu Lys Tyr
1$ 180 185 190
get cat gaa att atg act ccc aga act atg gtc ttt tca ctt cat gaa 624
Ala His Glu Ile Met Thr Pro Ar4 Thr Met Val Phe Ser Leu His Glu
195 200 205
aac ctt act gtc tct gaa get tat agc aac ccc aaa ata tgg aac tat 672
Asn Leu Thr Val Ser Glu Ala Tyr Ser Asn Pro Lys Ile Trp Asn Tyr
210 215 220
2$ agt cgc atc cct act tat gga gaa aat aac gaa gac att act ggc att 720
Ser Arg Ile Pro Thr Tyr Gly Glu Asn Asn Glu Asp Ile Thr Gly Ile
225 230 235 240
atc caa cga tat gaa att gga cga tat atg acc aat gga gaa aca gaa 768
Ile Gln Arg Tyr Glu Ile Gly Arg Tyr Met Thr Asn Gly Glu Thr Glu
245 250 255
aaa aaa ctt tta gaa att atg caa cca gca aaa ttt gtc ctt gaa agt 816
Lys Lys Leu Leu Glu Ile Met Gln Pro Ala Lys Phe Val Leu Glu Ser
3$ 260 265 270
caa act gta gat cat tta ctt ctt gca ttt tta gaa gaa aga caa cat 864
Gln Thr Val Asp His Leu Leu Leu Ala Phe Leu Glu Glu Arg Gln His
275 280 285
ctt ttt att gta ctt gat gag tat ggg gga tta tct ggt gtt gtt tcc 912
Leu Phe Ile Val Leu Asp Glu Tyr Gly Gly Leu Ser Gly Val Val Ser
290 295 300
4$ tta gaa gat gta tta gaa act atg ctt gga aga gaa att gtt gat gaa 960
Leu Glu Asp Val Leu Glu Thr Met Leu Gly Arg Glu Ile Val Asp Glu
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305 310 315 320
agt gat aca aca cct gat ctt aga gca ctt gca aaa aaa aga cat agt 1008
Ser Asp Thr Thr Pro Asp Leu Arg Ala Leu Ala Lys Lys Arg His Ser
325 330 335
gca tta atc caa aat aat aaa aat act ctt tta aaa taa 1047
Ala Leu Ile Gln Asn Asn Lys Asn Thr Leu Leu Lys
340 345
<210>
14
<211> 8
34
<212> T
PR
<213> wsonia
La intracellularis
<400>
14
Met IleIleLeuLeuGlyThrValPheLeuIleValLeuIleSerAla
1 5 10 15
Leu CysSerMetMetGluAlaAlaIleTyrSerIleProIleThrTyr
20 25 30
Ile GluHisLeuArgGluGlnGlySerLysLysGlyGluLysLeuTyr
35 40 45
Tyr LeuHisSerAsnIleAspGlnProIleThrAlaValLeuIleLeu
50 55 60
Asn ThrIleAlaAsnThrAlaGlyAlaAlaLeuAlaGlyAlaIleAla
65 70 75 80
Thr ThrThrLeuHisGluSerThrLysProPhePheAlaAlaIleLeu
85 90 95
Thr LeuLeuIleLeuAlaPheGlyGluIleIleProLysThrLeuGly
100 105 110
Val AlaTyrSerLysArgIleAlaIleIleLeuLeuAsnProLeuSer
115 120 125
Ile LeuIleValThrLeuLysProLeuIleMetLeuSerSerTyrLeu
130 135 140
Thr ArgLeuValSerProArgLysArgProThrValThrGluAspAsp
145 150 155 160
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Ile Arg Ala Leu Thr Ser Leu Ser Arg Glu Ser Gly Arg Ile Lys Pro
165 170 175
Tyr Glu Glu His Val Ile Lys Asn Ile Leu Ser Leu Asp Leu Lys Tyr
180 185 190
Ala His Glu Ile Met Thr Pro Arg Thr Met Val Phe Ser Leu His Glu
195 200 205
Asn Leu Thr Val Ser Glu Ala Tyr Ser Asn Pro Lys Ile Trp Asn Tyr
210 215 220
Ser Arg Ile Pro Thr Tyr Gly Glu Asn Asn Glu Asp Ile Thr Gly Ile
IS 225 230 235 240
Ile Gln Arg Tyr Glu Ile Gly Arg Tyr Met Thr Asn Gly Glu Thr Glu
245 250 255
Lys Lys Leu Leu Glu Ile Met Gln Pro Ala Lys Phe Val Leu Glu Ser
260 265 270
Gln Thr Val Asp His Leu Leu Leu Ala Phe Leu Glu Glu Arg Gln His
275 280 285
Leu Phe Ile Val Leu Asp Glu Tyr Gly Gly Leu Ser Gly Val Val Ser
290 295 300
Leu Glu Asp Val Leu Glu Thr Met Leu Gly Arg Glu Ile Val Asp Glu
305 310 315 320
Ser Asp Thr Thr Pro Asp Leu Arg Ala Leu Ala Lys Lys Arg His Ser
325 330 335
Ala Leu Ile Gln Asn Asn Lys Asn Thr Leu Leu Lys
340 345
<210> 15
<211> 1812
<212> DNA
<213> Lawsonia intracellularis
<220>
<221> CDS
<222> (1)..(1809)
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<400> 15
atg caa aaa gta tgt tat ttt ttt ctt ata acc ttt ttc tac ttt ttc 48
Met Gln Lys Val Cys Tyr Phe Phe Leu Ile Thr Phe Phe Tyr Phe Phe
1 5 10 15
ata aca gaa aat tat ctc ttt get aca tca att acc act tcc aca att 96
Ile Thr Glu Asn Tyr Leu Phe Ala Thr Ser Ile Thr Thr Ser Thr Ile
20 25 30
aac caa caa cat ata gca tat aca gtt act ttt acc tct cca gaa aat 144
Asn Gln Gln His Ile Ala Tyr Thr Val Thr Phe Thr Ser Pro Glu Asn
35 40 45
IS cct aat ctt gca aca gag atg gaa aca cat agt gaa tta gta aag ctt 192
Pro Asn Leu Ala Thr Glu Met Glu Thr His Ser Glu Leu Val Lys Leu
50 55 60
gca aat caa tct tta gat agt aaa ata ggt tta aat tta cgt gtt aaa 240
Ala Asn Gln Ser Leu Asp Ser Lys Ile Gly Leu Asn Leu Arg Val Lys
65 70 75 80
gaa gat ata agt aca gca caa aaa att ctt gac tcg aat ggt tat tat 288
Glu Asp Ile Ser Thr Ala Gln Lys Ile Leu Asp Ser Asn Gly Tyr Tyr
85 90 95
agt gga agt gtc gag gga aag att gac tgg cag acg aac cct att agt 336
Ser Gly Ser Val Glu Gly Lys Ile Asp Trp Gln Thr Asn Pro Ile Ser
100 105 110
atc caa atc caa ttt aaa cca aat gta caa tat aaa ata aat aca ata 384
Ile Gln Ile Gln Phe Lys Pro Asn Val Gln Tyr Lys Ile Asn Thr Ile
115 120 125
cat atc caa tac ctt gat agt gaa ctt gca tat ctc cct ctt tcc tta 432
His Ile Gln Tyr Leu Asp Ser Glu Leu Ala Tyr Leu Pro Leu Ser Leu
130 135 140
gaa gaa ttc aat cte tct aaa ggt aat ect get ett get gtt aat atc 480
Glu Glu Phe Asn Leu Ser Lys Gly Asn Pro Ala Leu Ala Val Asn Ile
145 150 155 160
cta tcc tct gta agt agc ctc atg caa tat ata cat aat aat gga tat 528
Leu Ser Ser Val Ser Ser Leu Met Gln Tyr Ile His Asn Asn Gly Tyr
16s 170 17s
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cca tta gcc aaa ata aaa aaa act caa tac ata att aat cgg atg gat 576
Pro Leu Ala Lys Ile Lys Lys Thr Gln Tyr Ile Ile Asn Arg Met Asp
180 185 190
$ tat aca ttt gat att gat tta gta ata aga caa gga ccg tta ctc cat 624
Tyr Thr Phe Asp Ile Asp Leu Val Ile Arg Gln Gly Pro Leu Leu His
195 200 205
atg ggt aaa gta caa cct caa cat aat ctc aat att tca aca ata ttc 672
Met Gly Lys Val Gln Pro Gln His Asn Leu Asn Ile Ser Thr Ile Phe
210 215 220
cta aat aaa att get aca tgg aag gaa gga agg gta tgg aac aat gca 720
Leu Asn Lys Ile Ala Thr Trp Lys Glu Gly Arg Val Trp Asn Asn Ala
IS 225 230 235 240
ctc ctt gat tct tat cga aca cgg ctt caa caa aca ggc ctt ttc agt 768
Leu Leu Asp Ser Tyr Ara Thr Arg Leu Gln Gln Thr Gly Leu Phe Ser
245 250 255
tct ata act ctc aat cca agg aat caa aaa gaa caa aat ggt aac acc 816
Ser Ile Thr Leu Asn Pro Arg Asn Gln Lys Glu Gln Asn Gly Asn Thr
260 265 270
tct ata gaa ctt gtt gca aca gaa gcc cct cca agg act att agt ggt 864
Ser Ile Glu Leu Val Ala Thr Glu Ala Pro Pro Arg Thr Ile Ser Gly
275 280 285
ggc tta caa tac tct tct gat caa ggt att ggt gca cgt ggg act tgg 912
Gly Leu Gln Tyr Ser Ser Asp Gln Gly Ile Gly Ala Arg Gly Thr Trp
290 295 300
gaa cat cga aat gtt ttt ggt aat gga gaa ctt ttt cgt ata aca gca 960
Glu His Arg Asn Val Phe Gly Asn Gly Glu Leu Phe Arg Ile Thr Ala
305 310 315 320
cca ata agt cga gat gat caa aaa att atg gca aac ttc caa aaa cca 1008
Pro Ile Ser Arg Asp Asp Gln Lys Ile Met Ala Asn Phe Gln Lys Pro
325 330 335
gcc ttt ggc cgt cca aat caa tca tta att agt gaa gca caa ctt aaa 1056
Ala Phe Gly Arg Pro Asn Gln Ser Leu Ile Ser Glu Ala Gln Leu Lys
340 345 350
aaa gaa aat aca aaa agt tac aaa caa caa ctt gca tct att get tta 1104
Lys Glu Asn Thr Lys Ser Tyr Lys Gln Gln Leu Ala Ser Ile Ala Leu
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355 360 365
gga att gaa cga caa ttt aat aga cgt tgg ttt ggt agt agc agt ctt 1152
Gly Ile Glu Arg Gln Phe Asn Arg Arg Trp Phe Gly Ser Ser Ser Leu
370 375 380
tca gtt gat aca gga ttt atg gat gat cga gat tct ata aaa aaa ata 1200
Ser Val Asp Thr Gly Phe Met Asp Asp Arg Asp Ser Ile Lys Lys Ile
385 390 395 400
ttt act ctt ttt ggc atc ccc tta tca ata aca agg gat agt tct aaa 1248
Phe Thr Leu Phe Gly Ile Pro Leu Ser Ile Thr Arg Asp Ser Ser Lys
405 410 415
IS gat cct ctt aat cct atc caa gga aca aaa get acc tta aat gtt act 1296
Asp Pro Leu Asn Pro Ile Gln Gly Thr Lys Ala Thr Leu Asn Val Thr
420 425 430
cct tat att ggt aaa tat aaa aaa aag att ttg act tta cgt agt cgg 1344
Pro Tyr Ile Gly Lys Tyr Lys Lys Lys Ile Leu Thr Leu Arg Ser Arg
435 440 445
ttt gat ttt agc ttt tac ata gac gtt ctt aaa aca ggg aaa ctt atc 1392
Phe Asp Phe Ser Phe Tyr Ile Asp Val Leu Lys Thr Gly Lys Leu Ile
450 455 460
ttg get aac aaa ata gca ata ggt tcc ctc cta ggg aaa gat ata gaa 1440
Leu Ala Asn Lys Ile Ala Ile Gly Ser Leu Leu Gly Lys Asp Ile Glu
465 470 475 480
aac tat cct gca ata cta agg ttt tat get ggg ggt ggt ggt agt gta 1488
Asn Tyr Pro Ala Ile Leu Arg Phe Tyr Ala Gly Gly Gly Gly Ser Val
485 490 495
aga ggg tat gac tat caa tca ttg gga cca aaa aat aaa tat ggg gat 1536
Arg Gly Tyr Asp Tyr Gln Ser Leu Gly Pro Lys Asn Lys Tyr Gly Asp
500 505 510
get att gga gga ctt tct ttt tca act att agt ttt gaa tta cga tta 1584
Ala Ile Gly Gly Leu Ser Phe Ser Thr Ile Ser Phe Glu Leu Arg Leu
515 520 525
aaa ata aca gaa tcc att ggc att gtg cca att tat tgg atg ggg gaa 1632
Lys Ile Thr Glu Ser Ile Gly Ile Val Pro Ile Tyr Trp Met Gly Glu
530 535 540
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tat tta cga aaa aaa aat ttc ctg act tta aaa aaa tca ata tat tgg 1680
Tyr Leu Arg Lys Lys Asn Phe Leu Thr Leu Lys Lys Ser Ile Tyr Trp
545 550 555 560
ggg gta ggc ctg ggg cta cga tat tat aca agt ttt gcc ccc ata cgt 1728
Gly Val Gly Leu Gly Leu Arg Tyr Tyr Thr Ser Phe Ala Pro Ile Arg
565 570 575
tta gat ata gca act cca ctt caa gat aga agc cat aat aaa cac ttt 1776
1~ Leu Asp Ile Ala Thr Pro.Leu Gln Asp Arg Ser His Asn Lys His Phe
580 585 590
caa ctt tat att agt att ggg caa gca ttc taa tga 1812
Gln Leu Tyr Ile Ser Ile Gly Gln Ala Phe
IS 595 600
<210> 16
<211> 602
<212> PRT
<213> Lawsonia intracellularis
<400> 16
Met Gln Lys Val Cys Tyr Phe Phe Leu Ile Thr Phe Phe Tyr Phe Phe
25 1 5 10 15
Ile Thr Glu Asn Tyr Leu Phe Ala Thr Ser Ile Thr Thr Ser Thr Ile
20 25 30
3~ Asn Gln Gln His Ile Ala Tyr Thr Val Thr Phe Thr Ser Pro Glu Asn
35 40 45
Pro Asn Leu Ala Thr Glu Met Glu Thr His Ser Glu Leu Val Lys Leu
50 55 60
Ala Asn Gln Ser Leu Asp Ser Lys Ile Gly Leu Asn Leu Arg Val Lys
65 70 75 80
Glu Asp Ile Ser Thr Ala Gln Lys Ile Leu Asp Ser Asn Gly Tyr Tyr
85 90 95
Ser Gly Ser Val Glu Gly Lys Ile Asp Trp Gln Thr Asn Pro Ile Ser
100 105 110
Ile Gln Ile Gln Phe Lys Pro Asn Val Gln Tyr Lys Ile Asn Thr Ile
115 120 125
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His IleGlnTyrLeuAspSerGluLeuAlaTyr LeuProLeuSerLeu
130 135 140
Glu GluPheAsnLeuSerLysGlyAsnProAla LeuAlaValAsnIle
145 150 155 160
Leu SerSerValSerSerLeuMetGlnTyrIle HisAsnAsnGlyTyr
165 170 175
Pro LeuAlaLysIleLysLysThrGlnTyrIle IleAsnArgMetAsp
180 185 190
Tyr ThrPheAspIleAspLeuValIleArgGln GlyProLeuLeuHis
195 200 205
Met GlyLysValGlnProGlnHisAsnLeuAsn IleSerThrIlePhe
210 215 220
Leu AsnLysIleAlaThrTrpLysGluGlyArg ValTrpAsnAsnAla
225 230 235 240
Leu LeuAspSerTyrArgThrArgLeuGlnGln ThrGlyLeuPheSer
245 250 255
Ser IleThrLeuAsnProArgAsnGlnLysGlu GlnAsnGlyAsnThr
260 265 270
Ser IleGluLeuValAlaThrGluAlaProPro ArgThrIleSerGly
275 280 285
Gly LeuGlnTyrSerSerAspGlnGlyIleGly AlaArgGlyThrTrp
290 295 300
Glu HisArgAsnValPheGlyAsnGlyGluLeu PheArgIleThrAla
305 310 315 320
Pro IleSerArgAspAspGlnLysIleMetAla AsnPheGlnLysPro
325 330 335
Ala PheGlyArgProAsnGlnSerLeuIleSer GluAlaGlnLeuLys
340 345 350
Lys GluAsnThrLysSerTyrLysGlnGlnLeu AlaSerIleAlaLeu
355 360 365
gaa cat cga aat gtt ttt ggt aat gga gaa ctt ttt cgt ata aca gca 960
Glu His Arg Asn Val Phe Gly
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Gly Ile Glu Arg Gln Phe Asn Arg Arg Trp Phe Gly Ser Ser Ser Leu
370 375 380
Ser Val Asp Thr Gly Phe Met Asp Asp Arg Asp Ser Ile Lys Lys Ile
385 390 395 400
Phe Thr Leu Phe Gly Ile Pro Leu Ser Ile Thr Arg Asp Ser Ser Lys
405 410 415
Asp Pro Leu Asn Pro Ile Gln Gly Thr Lys Ala Thr Leu Asn Val Thr
420 425 430
Pro Tyr Ile Gly Lys Tyr Lys Lys Lys Ile Leu Thr Leu Arg Ser Arg
435 440 445
Phe Asp Phe Ser Phe Tyr Ile Asp Val Leu Lys Thr Gly Lys Leu Ile
450 455 460
Leu Ala Asn Lys Ile Ala Ile Gly Ser Leu Leu Gly Lys Asp Ile Glu
465 470 475 480
Asn Tyr Pro Ala Ile Leu Arg Phe Tyr Ala Gly Gly Gly Gly Ser Val
485 490 495
Arg Gly Tyr Asp Tyr Gln Ser Leu Gly Pro Lys Asn Lys Tyr Gly Asp
500 505 510
Ala Ile Gly Gly Leu Ser Phe Ser Thr Ile Ser Phe Glu Leu Arg Leu
515 520 525
Lys Ile Thr Glu Ser Ile Gly Ile Val Pro Ile Tyr Trp Met Gly Glu
530 535 540
Tyr Leu Arg Lys Lys Asn Phe Leu Thr Leu Lys Lys Ser Ile Tyr Trp
545 550 555 560
Gly Val Gly Leu Gly Leu Arg Tyr Tyr Thr Ser Phe Ala Pro Ile Arg
565 570 575
Leu Asp Ile Ala Thr Pro Leu Gln Asp Arg Ser His Asn Lys His Phe
580 585 590
Gln Leu Tyr Ile Ser Ile Gly Gln Ala Phe
595 600
<zlo> 17
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<211> 4149
<212> DNA
<213> Lawsonia intracellularis
<220>
<221> CDS
<222> (1)..(4146)
<400> 17
1~ atg aat aac aca aaa ata ctt tct aag tta ctc tat acc ctc tta gga 48
Met Asn Asn Thr Lys Ile Leu Ser Lys Leu Leu Tyr Thr Leu Leu Gly
1 5 10 15
gca ttt acg tta ttt tta gga ctt att att aca ggc att ctt ttt ata 96
IS Ala Phe Thr Leu Phe Leu Gly Leu Ile Ile Thr Gly Ile Leu Phe Ile
20 25 30
cgg acc tct aca ggc att get tgg att aaa aat aca gtt tct tct tta 144
Arg Thr Ser Thr Gly Ile Ala Trp Iie Lys Asn Thr Val Ser Ser Leu
2~ 35 40 45
ctt caa caa caa gga att ata cta caa gta tct tca att att gga cca 192
Leu Gln Gln Gln Gly Ile Ile Leu Gln Val Ser Ser Ile Ile Gly Pro
50 55 60
ttc cca gaa caa att act att aat gaa ctt agc ctt agt gat gtg aat 240
Phe Pro Glu Gln Ile Thr Ile Asn Glu Leu Ser Leu Ser Asp Val Asn
65 70 75 8G
gga act tac ctt aca ata tct aac tta gaa atc caa tca aac tta tgg 288
Gly Thr Tyr Leu Thr Ile Ser Asn Leu Glu Ile Gln Ser Asn Leu Trp
85 90 95
get tta ttc aaa ggt caa ctt gaa att ctg tct ttt gaa ctt aat gat 336
Ala Leu Phe Lys Gly Gln Leu Glu Ile Leu Ser Phe Glu Leu Asn Asp
100 105 110
ctt gta tta tat cgc tta ccc tca aat aat aat cta aaa aaa tca tct 384
Leu Val Leu Tyr Arg Leu Pro Ser Asn Asn Asn Leu Lys Lys Ser Ser
4~ 115 120 125
aca agt ttt gtg tta cct cac ata tca ttt gat tta act cca tgg tgg 432
Thr Ser Phe Val Leu Pro His Ile Ser Phe Asp Leu Thr Pro Trp Trp
130 135 140
act gaa cat att cgt att caa aac atc cat att aac aat aca caa ctt 480
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Thr Glu His Ile Arg Ile Gln Asn Ile His Ile Asn Asn Thr Gln Leu
145 150 155 160
tcc tct gat att ata ggt att cca ttg gta tta tcc ctt gag ggt gat 528
Ser Ser Asp Ile Ile Gly Ile Pro Leu Val Leu Ser Leu Glu Gly Asp
165 170 175
ggt aca tta aca aat tgg aat gga aca ttt caa cta tcc tct tct aac 576
Gly Thr Leu Thr Asn Trp Asn Gly Thr Phe Gln Leu Ser Ser Ser Asn
180 185 190
aaa aca aaa att ata gga acg ctt cgt tac caa ggg aat aag aca caa 624
Lys Thr Lys Ile Ile Gly Thr Leu Arg Tyr Gln Gly Asn Lys Thr Gln
195 200 205
ttt ttt gaa tat gtt cat cct aca cgg ata gta aca cta gag ata gac 672
Phe Phe Glu Tyr Val His Pro Thr Arg Ile Val Thr Leu Glu Ile Asp
210 215 220
agc gta get gat aaa aag tca tat aat aat agt atc ctt gaa caa cct 720
Ser Val Ala Asp Lys Lys Ser Tyr Asn Asn Ser Ile Leu Glu Gln Pro
225 230 235 240
cta cat tta cac ctt tct att tat cct gaa cat aat aga att atc tta 768
Leu His Leu His Leu Ser Ile Tyr Pro Glu His Asn Arg Ile Ile Leu
245 250 255
cac tca tta cta get gaa tat ggt agc tgg tta ctt aca tca gaa agt 816
His Ser Leu Leu Ala Glu Tyr Gly Ser Trp Leu Leu Thr Ser Glu Ser
260 265 270
att gaa gta tct aat gag caa tta aaa gga aat att tta tta aaa tat 864
Ile Glu Val Ser Asn Glu Gln Leu Lys Gly Asn Ile Leu Leu Lys Tyr
275 280 285
aat gga gaa get act cat caa ctt cct ata aaa aaa ctt aac tca tca 912
Asn Gly Glu Ala Thr His Gln Leu Pro Ile Lys Lys Leu Asn Ser Ser
290 295 300
att acc ctc agt ggc tca cta aat aaa cct aat ttt agt ata caa atg 960
Ile Thr Leu Ser Gly Ser Leu Asn Lys Pro Asn Phe Ser Ile Gln Met
305 310 315 320
aca tta cct gaa att aac att aca aaa aac ata ata gat ctt caa aca 1008
Thr Leu Pro Glu Ile Asn Ile Thr Lys Asn Ile Ile Asp Leu Gln Thr
325 330 335
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gaa ctt gtt att aat cta gga ctt ttc tct act cac tct gat att ctt 1056
Glu Leu Val Ile Asn Leu Gly Leu Phe Ser Thr His Ser Asp Ile Leu
340 345 350
aca tct ggg aca att aca gta cag gga gaa act ata ccc aat agt att 1104
Thr Ser Gly Thr Ile Thr Val Gln Gly Glu Thr Ile Pro Asn Ser Ile
355 360 365
1~ ctt tcc agt gca gtt gat ata ata gcc tct aca aca aca cat aca att 1152
Leu Ser Ser Ala Val Asp Ile Ile Ala Ser Thr Thr Thr His Thr Ile
370 375 380
acc tta gag cat gca acc tta aca tct cca gaa atg cat ttt tcc cta 1200
Thr Leu Glu His Ala Thr Leu Thr Ser Pro Glu Met His Phe Ser Leu
385 390 395 400
tct gga gaa ttt aat agt ctt cta gga aat atc gat gca aac cta aaa 1248
Ser Gly Glu Phe Asn Ser Leu Leu Gly Asn Ile Asp Ala Asn Leu Lys
405 410 415
ggt aat act cca act ctt agt ata ttt tct tct ctt ctt gga cta cct 1296
Gly Asn Thr Pro Thr Leu Ser Ile Phe Ser Ser Leu Leu Gly Leu Pro
420 425 430
gat ctt act ggg caa agt aac att act ata gga tta cac cgt caa ggg 1344
Asp Leu Thr Gly Gln Ser Asn Ile Thr Ile Gly Leu His Arg Gln Gly
435 440 445
tct tcc tct tca ata gaa gga aca gca act gtc tca ctt aat aat atg 1392
Ser Ser Ser Ser Ile Glu Gly Thr Ala Thr Val Ser Leu Asn Asn Met
450 455 460
aac tgg gga gta caa gca tta cag ggg aca tta ggt gat aat gca act 1440
Asn Trp Gly Val Gln Ala Leu Gln Gly Thr Leu Gly Asp Asn Ala Thr
465 470 475 480
cta agt gga ata tat aat tta act ccc ata gac tgg tct att tct tta 1488
Leu Ser Gly Ile Tyr Asn Leu Thr Pro Ile Asp Trp Ser Ile Ser Leu
4~ 485 490 495
aac aaa ttg aaa tta aca gca aag aat gtt tat get gaa ggc ctt att 1536
Asn Lys Leu Lys Leu Thr Ala Lys Asn Val Tyr Ala Glu Gly Leu Ile
500 505 510
aat ttt caa aaa aaa tac ata gat agc tct ata aat ctt ata att cct 1584
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Asn Phe Gln Lys Lys Tyr Ile Asp Ser Ser Ile Asn Leu Ile Ile Pro
515 520 525
aac ctt cag cta ata get cct cct ata ~tct gga gag tta caa tcc tta 1632
Asn Leu Gln Leu Ile Ala Pro Pro Ile Ser Gly Glu Leu Gln Ser Leu
530 535 540
att aca gtg tct gga aaa ctt gac gca cct tct ata gaa agc aaa att 1680
Ile Thr Val Ser Gly Lys Leu Asp Ala Pro Ser Ile Glu Ser Lys Ile
1~ 545 550 555 560
ttt tca tca caa ctc acc tgg aat gcg ctc caa ctt aat aat cct caa 1728
Phe Ser Ser Gln Leu Thr Trp Asn Ala Leu Gln Leu Asn Asn Pro Gln
565 570 575
IS
ctc ata ata act act act caa tct tct tcc tct gcg att aaa ggt aat 1776
Leu Ile Ile Thr Thr Thr Gln Ser Ser Ser Ser Ala Ile Lys Gly Asn
580 585 590
2~ ata aca ctc tcg get gag cca get tca tct gag gca tta acc ttt tca 1824
Ile Thr Leu Ser Ala Glu Pro Ala Ser Ser Glu Ala Leu Thr Phe Ser
595 600 605
agt aat tgg gga atc cta cct acg gaa ata cta gta gaa aaa att ata 1872
25 Ser Asn Trp Gly Ile Leu Pro Thr Glu Ile Leu Val Glu Lys Ile Ile
610 615 620
gga aat ata tta gga gta aat ctt gat ggt aat att aaa ata aca aaa 1920
Gly Asn Ile Leu Gly Val Asn Leu Asp Gly Asn Ile Lys Ile Thr Lys
3~ 625 630 635 640
aaa gat tac ctt ata aat ggt gat att att gca gaa gtt cag tct tgg 1968
Lys Asp Tyr Leu Ile Asn Gly Asp Ile Ile Ala Glu Val Gln Ser Trp
645 650 655
aaa gat att gca aac ata ttg caa ata cct att aga ggt tca gca tca 2016
Lys Asp Ile Ala Asn Ile Leu Gln Ile Pro Ile Arg Gly Ser Ala Ser
660 665 670
ata aaa ata cag ttt gat cca aag aat caa caa tgt att tct act caa 2064
Ile Lys Ile Gln Phe Asp Pro Lys Asn Gln Gln Cys Ile Ser Thr Gln
675 680 685
tgg caa tta aaa aat ttc ata tta ggt aat aat ttt aat gta act act 2112
Trp Gln Leu Lys Asn Phe Ile Leu Gly Asn Asn Phe Asn Val Thr Thr
690 695 700
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ata aaa gga aga gca gat aca ata caa ctt cat aag aat cct aca att 2160
Ile Lys Gly Arg Ala Asp Thr Ile Gln Leu His Lys Asn Pro Thr Ile
705 710 715 720
get ctc tct tca aaa att ggt get ggt aca tat gaa gac ttt caa tgg 2208
Ala Leu Ser Ser Lys Ile Gly Ala Gly Thr Tyr Glu Asp Phe Gln Trp
725 730 735
aca caa ggg acg tta gac ata aaa ggc aca tta aaa aat ttt aat agt 2256
Thr Gln Gly Thr Leu Asp Ile Lys Gly Thr Leu Lys Asn Phe Asn Ser
740 745 750
aaa ata aat ata gca gga caa aca act gta aac gca aac ttt caa aca 2304
1$ Lys Ile Asn Ile Ala Gly Gln Thr Thr Val Asn Ala Asn Phe Gln Thr
755 760 765
aat ctt ttt gaa aaa aat att aat ata act act ctt aat tta aaa aat 2352
Asn Leu Phe Glu Lys Asn Ile Asn Ile Thr Thr Leu Asn Leu Lys Asn
770 775 7ao
att caa aaa aat ata gga att aag ctc ctt cag cca ata aaa att ata 2400
Ile Gln Lys Asn Ile Gly Ile Lys Leu Leu Gln Pro Ile Lys Ile Ile
785 790 795 S00
gtc tca cct caa caa ttt gtt ctt aat aac tgt tca cta gca att ctt 2448
Val Ser Pro Gln Gln Phe Val Leu Asn Asn Cys Ser Leu Ala Ile Leu
805 810 B15
cca tct gga aca att aca act gat ata tat gtt act cct caa cga ctt 2496
Pro Ser Gly Thr Ile Thr Thr Asp Ile Tyr Val Thr Pro Gln Arg Leu
820 825 830
aat get aat gca atc att aaa gaa gtt tca ctt ctc tct ttc caa cca 2544
Asn Ala Asn Ala Ile Ile Lvs Glu Val Ser Leu Leu Ser Phe Gln Pro
835 840 845
ttt agt ata ctt ctt cct caa gga aat ata aat gga cac ata aca ctt 2592
Phe Ser Ile Leu Leu Pro Gln Gly Asn Ile Asn Gly His Ile Thr Leu
sso ass a6o
aca gga ata cct agt aaa cct aaa gga aca ctc tca ttt gat att cta 2640
Thr Gly Ile Pro Ser Lys Pro Lys Gly Thr Leu Ser Phe Asp Ile Leu
865 870 875 880
aac ata cat tat cca agg cca aat cca tca ata gca aac tta cat gta 2688
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Asn Ile His Tyr Pro Arg Pro Asn Pro Ser Ile Ala Asn Leu His Val
885 890 895
gaa ggg gaa att ata tct tct cct aac aat ata tgt aaa ctt aat gca 2736
Glu Gly Glu Ile Ile Ser Ser Pro Asn Asn Ile Cys Lys Leu Asn Ala
900 905 910
acc cta aca gaa aaa aaa gag cct ata cct ata tca ata caa gca aca 2784
Thr Leu Thr Glu Lys Lys Glu Pro Ile Pro Ile Ser Ile Gln Ala Thr
1~ 915 920 925
ctc cct ttt gag ttc aca gaa aac aat atc cct atg cta tct aaa atg 2832
Leu Pro Phe Glu Phe Thr Glu Asn Asn Ile Pro Met Leu Ser Lys Met
930 935 940
agg cct ttt tct gcc cat atc aag tgg act gga ata tta gat aca ctt 2880
Arg Pro Phe Ser Ala His Ile Lys Trp Thr Gly Ile Leu Asp Thr Leu
945 950 955 960
tgg aaa ctc att cca ctt act gat tac att atg get ggg aat gga tct 2928
Trp Lys Leu Ile Pro Leu Thr Asp Tyr Ile Met Ala Gly Asn Gly Ser
965 970 975
tta gat get tct ctt tct ggg act tta gat agt cca aca tat gca att 2976
Leu Asp Ala Ser Leu Ser Gly Thr Leu Asp Ser Pro Thr Tyr Ala Ile
980 985 990
ata aca aca ctt tct aat get aac ttt caa gat ctc tcc ctt ggt ctt 3024
Ile Thr Thr Leu Ser Asn Ala Asn Phe Gln Asp Leu Ser Leu Gly Leu
995 1000 1005
tac tta gaa aat atc aat get aaa tta cag gtc ttt tct aat aga atc 3072
Tyr Leu Glu Asn Ile Asn Ala Lys Leu Gln Val Phe Ser Asn Arg Ile
1010 1015 1020
tcc cat att caa get aca gca tct gat ggt aaa caa ggt agt ata caa 3120
Ser His Ile Gln Ala Thr Ala Ser Asp Gly Lys Gln Gly Ser Ile Gln
1025 1030 1035 1040
4~ ctt att ggt aat att ggc tca tct aaa gaa cac ttt cct ttg tct att 3168
Leu Ile Gly Asn Ile Gly Ser Ser Lys Glu His Phe Pro Leu Ser Ile
1045 1050 1055
aat ggc tcc ttt aca aac ctt get cca tta caa cgt aaa gac cta agt 3216
Asn Gly Ser Phe Thr Asn Leu Ala Pro Leu Gln Arg Lys Asp Leu Ser
1060 1065 1070
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ctt aca ctt tca gga gca get act ctt gaa gga aca tta aaa cag tct 3264
Leu Thr Leu Ser Gly Ala Ala Thr Leu Glu Gly Thr Leu Lys Gln Ser
1075 1080 1085
gaa gtt aaa ggc gat att gtt.att aac caa ggc gaa ttt caa ctt act 3312
Glu Val Lys Gly Asp Ile Val Ile Asn Gln Gly Glu Phe Gln Leu Thr
1090 1095 1100
gaa ggg tta acc agt aat att cca act ctt aat gta gtt gat agc act 3360
Glu Gly Leu Thr Ser Asn Ile Pro Thr Leu Asn Val Val Asp Ser Thr
1105 1110 1115 1120
caa caa caa aat aca aag acc aaa aaa get acc tat caa caa cct acc 3408
Gln Gln Gln Asn Thr Lys Thr Lys Lys Ala Thr Tyr Gln Gln Pro Thr
1125 1130 1135
tta tct att gcg tta agt atc ccg aat cgt ttt ttt gtc cgt agt agt 3456
Leu Ser Ile Ala Leu Ser Ile Pro Asn Arg Phe Phe Val Arg Ser Ser
1140 1145 1150
atg ttt gaa agt gag tgg gga ggg aac cta act att aac aaa gtc ata 3504
Met Phe Glu Ser Glu Trp Gly Gly Asn Leu Thr Ile Asn Lys Val Ile
1155 1160 1165
aca agt cct gtt att aca gga gca cta act tct ata aga gga aat ttt 3552
Thr Ser Pro Val Ile Thr Gly Ala Leu Thr Ser Ile Arg Gly Asn Phe
1170 1175 1180
aat tta cta gga aaa caa ttt tct ctt get aaa agt aca ata tca ttt 3600
Asn Leu Leu Gly Lys Gln Phe Ser Leu Ala Lys Ser Thr Ile Ser Phe
1185 1190 1195 1200
cca gga tca gtt cca cca aac cca cta ctc aat att tct tta aca tat 3648
Ser Gly Ser Val Pro Pro Asn Pro Leu Leu Asn Ile Ser Leu Thr Tyr
1205 1210 1215
tca tca cct tct att aca get ata ggc att att aaa ggt aca act agt 3696
Ser Ser Pro Ser Ile Thr Ala Ile Gly Ile Ile Lys Gly Thr Thr Ser
4~ 1220 1225 1230
aat cct aat att act ttt tca agt aca cca cct tta cct caa gat gaa 3744
Asn Pro Asn Ile Thr Phe Ser Ser Thr Pro Pro Leu Pro Gln Asp Glu
1235 1240 1245
ata gtt tcc caa gtt ctt ttt ggt aaa agc tca caa agt ctt agc agg 3792
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Ile Val Ser Gln Val Leu Phe Gly Lys Ser Ser Gln Ser Leu Ser Arg
1250 1255 1260
ata caa~gcc ata caa ctt get caa gaa tta gca aac tta aca gga ttt 3840
Ile Gln Ala Ile Gln Leu Ala Gln Glu Leu Ala Asn Leu Thr Gly Phe
1265 1270 1275 1280
aat act gga agt atg aat ttc cta aca aat att cga cag aca tta ggg 3888
Asn Thr Gly Ser Met Asn Phe Leu Thr Asn Ile Arg Gln Thr Leu Gly
1~ 1285 1290 1295
tta gat ata ctt agc tta ggg aca act tct aat aga aaa gcc aat aca 3936
Leu Asp Ile Leu Ser Leu Gly Thr Thr Ser Asn Arg Lys Ala Asn Thr
1300 1305 1310
IS
tcc aac tca aac gat caa ata gaa gat atc cct gtt ata gaa cta ggt 3984
Ser Asn Ser Asn Asp Gln Ile Glu Asp Ile Pro Val Ile Glu Leu Gly
1315 1320 1325
aaa tat att aca gac act gtt tat gtt ggt gtt gaa caa agt tat tta 4032
Lys Tyr Ile Thr Asp Thr Val Tyr Val Gly Val Glu Gln Ser Tyr Leu
1330 1335 1340
gat agt aat gat act ggg gca aga ata tca gtt gaa ctt gca cct aat 4080
25 Asp Ser Asn Asp Thr Gly Ala Arg Ile Ser Val Glu Leu Ala Pro Asn
1345 1350 1355 1360
ttt aat ctt gaa ggt aga aca ggg act caa tat agt gag ata ggt att 4128
Phe Asn Leu Glu Gly Arg Thr Gly Thr Gln Tyr Ser Glu Ile Gly Ile
3~ 1365 1370 1375
aat tgg aaa aaa gat tat taa 4149
Asn Trp Lys Lys Asp Tyr
1380
<zlo> is
<z11> 1382
<212> PRT
<213> Lawsonia intracellularis
<400> 18
Met Asn Asn Thr Lys Ile Leu Ser Lys Leu Leu Tyr Thr Leu Leu Gly
1 5 10 15
Ala Phe Thr Leu Phe Leu Gly Leu Ile Ile Thr Gly Ile Leu Phe Ile
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20 25 30
Arg ThrSerThrGlyIleAlaTrpIleLysAsnThrValSerSerLeu
35 40 45
Leu GlnGlnGlnGlyIleIleLeuGlnValSerSerIleIleGlyPro
50 55 60
Phe ProGluGlnIleThrIleAsnGluLeuSerLeuSerAspValAsn
65 70 75 80
Gly ThrTyrLeuThrIleSerAsnLeuGluIleGlnSerAsnLeuTrp
85 90 95
IS Ala LeuPheLysGlyGlnLeuGluIleLeuSerPheGluLeuAsnAsp
100 105 110
Leu ValLeuTyrArgLeuProSerAsnAsnAsnLeuLysLysSerSer
115 120 125
Thr SerPheValLeuProHisIleSerPheAspLeuThrProTrpTrp
130 135 140
Thr GluHisIleArgIleGlnAsnIleHisIleAsnAsnThrGlnLeu
145 150 155 160
Ser SerAspIleIleGlyIleProLeuValLeuSerLeuGluGlyAsp
165 170 175
Gly ThrLeuThrAsnTrpAsnGlyThrPheGlnLeuSerSerSerAsn
180 185 190
Lys ThrLysIleIleGlyThrLeuArgTyrGlnGlyAsnLysThrGln
195 200 205
Phe PheGluTyrValHisProThrArgIleValThrLeuGluIleAsp
210 215 220
Ser ValAlaAspLysLysSerTyrAsnAsnSerIleLeuGluGlnPro
225 230 235 240
Leu HisLeuHisLeuSerIleTyrProGluHisAsnArgIleIleLeu
245 250 255
His SerLeuLeuAlaGluTyrGlySerTrpLeuLeuThrSerGluSer
260 265 270
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Ile GluValSerAsnGluGlnLeuLysGlyAsn IleLeuLeu LysTyr
275 280 285
Asn GlyGluAlaThrHisGlnLeuProIleLys LysLeuAsn SerSer
290 295 300
Ile ThrLeuSerGlySerLeuAsnLysProAsn PheSerIle GlnMet
305 310 315 320
Thr LeuProGluIleAsnIleThrLysAsnIle IleAspLeu GlnThr
325 330 335
Glu LeuValIleAsnLeuGlyLeuPheSerThr HisSerAsp IleLeu
IS 340 345 350
Thr SerGlyThrIleThrValGlnGlyGluThr IleProAsn SerIle
355 360 365
Leu SerSerAlaValAspIleIleAlaSerThr ThrThrHis ThrIle
370 375 380
Thr LeuGluHisAlaThrLeuThrSerProGlu MetHisPhe SerLeu
385 390 395 400
Ser GlyGluPheAsnSerLeuLeuGlyAsnIle AspAlaAsn LeuLys
405 410 415
Gly AsnThrProThrLeuSerIlePheSerSer LeuLeuGly LeuPro
420 425 430
Asp LeuThrGlyGlnSerAsnIleThrIleGly LeuHisArg GlnGly
435 440 445
Ser SerSerSerIleGluGlyThrAlaThrVal SerLeuAsn AsnMet
450 455 460
Asn TrpGlyValGlnAlaLeuGlnGlyThrLeu GlyAspAsn AlaThr
465 470 475 480
Leu SerGlyIleTyrAsnLeuThrProIleAsp TrpSerIle SerLeu
485 490 495
Asn LysLeuLysLeuThrAlaLysAsnValTyr AlaGluGly LeuIle
500 505 510
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Asn PheGlnLysLysTyrIleAspSerSerIleAsnLeuIleIlePro
515 520_ 525
Asn LeuGlnLeuIleAlaProProIleSerGlyGluLeuGlnSerLeu
530 535 540
Ile ThrValSerGlyLysLeuAspAlaProSerIleGluSerLysIle
545 550 555 560
Phe SerSerGlnLeuThrTrpAsnAlaLeuGlnLeuAsnAsnProGln
565 570 575
Leu IleIleThrThrThrGlnSerSerSerSerAlaIleLysGlyAsn
580 585 590
Ile ThrLeuSerAlaGluProAlaSerSerGluAlaLeuThrPheSer
595 600 605
Ser AsnTrpGlyIleLeuProThrGluIleLeuValGluLysIleIle
610 615 620
Gly AsnIleLeuGlyValAsnLeuAspGlyAsnIleLysIleThrLys
625 630 635 640
Lys AspTyrLeuIleAsnGlyAspIleIleAlaGluValGlnSerTrp
645 650 655
Lys AspIleAlaAsnIleLeuGlnIleProIleArgGlySerAlaSer
660 665 670
Ile LysIleGlnPheAspProLysAsnGlnGlnCysIleSerThrGln
675 680 685
Trp GlnLeuLysAsnPheIleLeuGlyAsnAsnPheAsnValThrThr
690 695 700
Ile LysGlyArgAlaAspThrIleGlnLeuHisLysAsnProThrIle
705 710 715 720
Ala LeuSerSerLysIleGlyAlaGlyThrTyrGluAspPheGlnTrp
725 730 735
Thr GlnGlyThrLeuAspIleLysGlyThrLeuLysAsnPheAsnSer
740 745 750
Lys IleAsnIleAlaGlyGlnThrThrValAsnAlaAsnPheGlnThr
CA 02399276 2002-08-02
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755 760 765
Asn Leu Phe Glu Lys Asn Ile Asn Ile Thr Thr Leu Asn Leu Lys Asn
770 775 780
Ile Gln Lys Asn Ile Gly Ile Lys Leu Leu Gln Pro Ile Lys Ile Ile
785 790 795 800
Val Ser Pro Gln Gln Phe Val Leu Asn Asn Cys Ser Leu Ala Ile Leu
805 810 815
Pro Ser Gly Thr Ile Thr Thr Asp Ile Tyr Val Thr Pro Gln Arg Leu
820 825 830
IS Asn Ala Asn Ala Ile Ile Lys Glu Val Ser Leu Leu Ser Phe Gln Pro
835 840 845
Phe Ser Ile Leu Leu Pro Gln Gly Asn Ile Asn Gly His Ile Thr Leu
850 855 860
Thr Gly Ile Pro Ser Lys Pro Lys Gly Thr Leu Ser Phe Asp Ile Leu
865 870 875 880
Asn Ile His Tyr Pro Arg Pro Asn Pro Ser Ile Ala Asn Leu His Val
885 890 895
Glu Gly Glu Ile Ile Ser Ser Pro Asn Asn Ile Cys Lys Leu Asn Ala
900 905 910
Thr Leu Thr Glu Lys Lys Glu Pro Ile Pro Ile Ser Ile Gln Ala Thr
915 920 925
Leu Pro Phe Glu Phe Thr Glu Asn Asn Ile Pro Met Leu Ser Lys Met
930 935 940
Arg Pro Phe Ser Ala His Ile Lys Trp Thr Gly Ile Leu Asp Thr Leu
945 950 955 960
Trp Lys Leu Ile Pro Leu Thr Asp Tyr Ile Met Ala Gly Asn Gly Ser
965 970 975
Leu Asp Ala Ser Leu Ser Gly Thr Leu Asp Ser Pro Thr Tyr Ala Ile
980 985 990
Ile Thr Thr Leu Ser Asn Ala Asn Phe Gln Asp Leu Ser Leu Gly Leu
995 1000 1005
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Tyr LeuGluAsnIleAsnAlaLysLeuGlnValPheSerAsnArgIle
1 010 1015 1 020
Ser HisIleGlnAlaThrAlaSerAspGlyLysGlnGlySerIleGln
1025 1030 1035 1 040
Leu IleGlyAsnIleGlySerSerLysGluHisPheProLeuSerIle
1 045 1050 1055
Asn GlySerPheThrAsnLeuAlaProLeuGlnArgLysAspLeuSer
1060 1065 1070
Leu ThrLeuSerGlyAlaAlaThrLeuGluGlyThrLeuLysGlnSer
1075 1080 1085
Glu ValLysGlyAspIleValIleAsnGlnGlyGluPheGlnLeuThr
1090 1095 1100
Glu GlyLeuThrSerAsnIleProThrLeuAsnValValAspSerThr
1105 1110 1115 1120
Gln GlnGlnAsnThrLysThrLysLysAlaThrTyrGlnGlnProThr
1125 1130 1135
Leu SerIleAlaLeuSerIleProAsnArgPhePheValArgSerSer
1140 1145 1150
Met PheGluSerGluTrpGlyGlyAsnLeuThrIleAsnLysValIle
1155 1160 1165
Thr SerProValIleThrGlyAlaLeuThrSerIleArgGlyAsnPhe
1170 1175 1180
Asn LeuLeuGlyLysGlnPheSerLeuAlaLysSerThrIleSerPhe
1185 1190 1195 1200
Ser GlySerValProProAsnProLeuLeuAsnIleSerLeuThrTyr
1205 1210 1215
Ser SerProSerIleThrAlaIleGlyIleIleLysGlyThrThrSer
1220 1225 1230
Asn ProAsnIleThrPheSerSerThrProProLeuProGlnAspGlu
1235 1240 1245
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-42-
Ile Val Ser Gln Val Leu Phe Gly Lys Ser Ser Gln Ser Leu Ser Arg
1250 1255 1260
Ile Gln Ala Ile Gln Leu Ala Gln Glu Leu Ala Asn Leu Thr Gly Phe
1265 1270 1275 1280
Asn Thr Gly Ser Met Asn Phe Leu Thr Asn Ile Arg Gln Thr Leu Gly
1285 1290 1295
1~ Leu Asp Ile Leu Ser Leu Gly Thr Thr Ser Asn Arg Lys Ala Asn Thr
1300 1305 1310
Ser Asn Ser Asn Asp Gln Ile Glu Asp Ile Pro Val Ile Glu Leu Gly
1315 1320 1325
Lys Tyr Ile Thr Asp Thr Val Tyr Val Gly Val Glu Gln Ser Tyr Leu
1330 1335 1340
Asp Ser Asn Asp Thr Gly Ala Arg Ile Ser Val Glu Leu Ala Pro Asn
2~ 1345 1350 1355 1360
Phe Asn Leu Glu Gly Arg Thr Gly Thr Gln Tyr Ser Glu Ile Gly Ile
1365 1370 1375
Asn Trp Lys Lys Asp Tyr
1380
<210> 19
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 19
catattcaag gtacagcatc tgatgg 26
4~ <210> 20
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
- 43 -
Oligonucleotide probe/primer
<400> 20
ctcctttaca aaccttgctc c 21
<z1o> 21
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
1$
<400> 21
gctcatctaa agaacacttt cc 22
<zlo> zz
<211> 23
<212> DNA
<213> Artificial Sequence
2$ <220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 22
caaggtagta tacaacttat tgg 23
<210> 23
<211> 23
3$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 23
gacctaagtc ttacactttc agg 23
4$
<210> 24
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-44-
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 24
gtattaatac tacattagtt gacg 24
<210> 25
<211> 21
IS <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 25
ggataataat ggaaaaagtg g 21
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 26
caagcaatgc ctgtagaggt cc 22
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
- 4$ -
<400> 27
aagaatgcct gtaataataa gtcc 24
<210> 28
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
1$ <400> 28
ttggggaatc ctacctacg 19
<210> 29
<z11> 21
<212> DNA
<213> Artificial Sequence
<220>
2$ <223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 29
tattaggagt aaatcttgat g 21
<210> 30
<211> 22
<212> DNA
3$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 30
gcaggacaaa caactgtaaa cg 22
4$ <zlo> 31
<211> zz
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-46-
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide. probe/primer
<400> 31
gaggaagaag tatactaaat gg 22
<210> 32
<211> 20
c212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 32
tgttggacta tctaaagtcc 20
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence
<2zo>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 33
ctattgatgg atttggcctt gg 22
<210> 34
<211> 20 <212>
DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-47-
<400> 34
gtgctggtac atatgaagac 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 35
ttcatcacct tctattacag 20
<210> 36
<211> 20
2,0 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 36
ggaaactatt tcatcttgag 20
<210> 37
<211> zo
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 37
attaggtgca agttcaactg 20
<210> 38 '
<211> 20
<212> DNA
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
- 48 -
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
$ Oligonucleotide probe/primer
<400> 38
tttagatagt aatgatactg 20
1~
<210> 39
<211> 36
<212> DNA
<213> Artificial Sequence
1$
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 39
ttattatatt atgttttttg taatgttaat ttcagg 36
<210> 40
2$ <211> 28
<212> DNA
<213> Artificial Sequence
<220>
3~ <223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 40
gacatatgaa taacacaaaa atactttc 28
3$
<210> 41
<211> 41
<212> DNA
4~ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
4$
<400> 41
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-49-
gaggatcctc tagagttaat caaactgtat ttttattgat g 41
<210> 42
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
1~ <223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 42
gacatatgcg gacctctaca ggcattgctt g 31
<210> 43
<211> 24
<212> DNA
2~ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 43
gatcaggtag tccaagaaga gaag 24
3~ <210> 44
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 44
ttggaggatc ctctagagtt atcaggttgt aattgttcca gatgg 45
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-50-
<220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 45
ccttggttaa taacaatatc g 21
<zlo> 46
<z11> z3
<212> DNA
<213> Artificial Sequence
1$ <220>
<223> Description of Artificial Sequence:
Oligonucleotide probe/primer
<400> 46
caactccact tcaagataga agc 23
<210> 47
<211> 29
<212> DNA
2$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo
<400> 47
gaccatggaa aaagtatgtt atttttttc 29
<210> 48
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo
<400> 48
gaggatcctc tagagttaga atgcttgccc aatact 36
<210> 49
<211> 39
<212> DNA
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
- $1 -
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo
$
<400> 49
ttggaggatc ctctagagtt agaatgcttg cccaatact 39
<210> 50
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
1$ <223> Description of Artificial Sequence: oligo
<400> 50
ttgaccatgg ctacatcaat taccacttcc ac 32
<210> 51
<211> 29
<212> DNA
<213> Artificial Sequence
2$ <220>
<223> Description of Artificial Sequence: oligo
<400> 51
ggacatatga ataacacaaa aatactttc 29
<210> 52
<211> 44
<212> DNA
<213> Artificial Sequence
3$
<220>
<223> Description of Artificial Sequence: oligo
<400> 52
ttggaggatc ctctagagtt aatcaaactg tatttttatt gatg 44
<210> 53
<211> 32
<212> DNA
4$ <213> Artificial Sequence
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-$2-
<zzo>
<223> Description of Artificial Sequence: oligo
<400> 53
$ ggacatatgc ggacctctac aggcattgct tg 32
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo
1$ <400> 54
tgaggattat taagttggag 20
<210> 55
<211> zo
<212> DNA
<213> Artificial Sequence
<220>
2$ <223> Description of Artificial Sequence: oligo
<400> 55
gcatgcaacc ttaacatctc 20
<210> s6
<211> 20
<212> DNA
<213> Artificial Sequence
3$ <220>
<223> Description of Artificial Sequence: oligo
<400> 56
tttctgatgt aagtaaccag 20
<210> 57
<211> 20
<212> DNA
<213> Artificial Sequence
4$
<220>
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-$3-
<223> Description of Artificial Sequence: oligo
<400> 57
tctgcccata tcaagtggac 20
$
<210> 58
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo
<400> 58
1$ ggaacatttc aactatcctc 20
<210> 59
<211> 2°
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo
<400> 59
2$ gtaaggtaag ttccattcac 20
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo
3$ <400> 60
caacgtggat ccgaattcaa gcttc 25
<210> 61
<z11> 17
<212> PRT
<213> Artificial Sequence
<220>
4$ <223> Description of Artificial Sequence: Peptide
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-54-
<400> 61
Met Gly Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Ala Leu Leu Thr
1 5 10 15
Met
<210> 62
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
IS <223> Description of Artificial Sequence: Peptide
<400> 62
Ala Thr Ser Ile Thr Thr Ser
1 5
<210> 63
<211> 45
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Peptide
<400> 63
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
35 40 45
<210> 64
<211> 50
<212> PRT
<213> Artificial Sequence
<220>
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-55-
<223> Description of Artificial Sequence: Peptide
<400> 64
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser
15
<210> 65
<211> 7
<212> PRT
20 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Peptide
25 <400> 65
Glu Phe Asn Leu Ser Lys Gly
1 5
30 <210> 66
<211> 17
<212> PRT
<213> Artificial Sequence
35 <z2o>
<223> Description of Artificial Sequence: Peptide
<400> 66
Met Gly Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Ala Leu Gly His
40 1 s to is
Met
<210> 67
CA 02399276 2002-08-02
WO 02/38594 PCT/AU01/01462
-56-
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Peptide
<400> 67
Arg Thr Ser Thr Gly Ile Ala
1 s
<210> sa
<211> 21
IS <212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Peptide
<400> 68
Asp Pro Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His
1 5 10 15
2S His His His His His
