Note: Descriptions are shown in the official language in which they were submitted.
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VACCINE COMPOSITIONS COMPRISING THE HELICOBACTER PYLORI FIgE POLYPEPTIDE
TECHNICAL FIELD
The present invention relates to polypeptides and vaccine compositions for
inducing a protective immune response to Helicobacter pylori infection. The
invention furthermore relates to the use of Helicobacter pylori polypeptides
in the
manufacture of compositions for the treatment or prophylaxis of Helicobacter
pylori
infection.
to
BACKGROUND ART
Helicobacter pylori
The gram-negative bacterium Helicobacter pylori (H. pylori) is an important
human
pathogen, involved in several gastroduodenal diseases. Colonization of gastric
epithelium by the bacterium leads to active inflammation and progressive
chronic
gastritis, with a greatly enhanced risk of progression to peptic ulcer
disease. A
lifelong inflammation of the gastric mucosa is very closely correlated with a
significantly enhanced risk for gastric cancer.
In order to colonize the gastric mucosa, H, pylori uses a number of virulence
factors. Such virulence factors comprise several adhesins, with which the
as bacterium associates with the mucus and/or binds to epithelial cells;
unease which
helps to neutralize the acid environment; and proteolytic enzymes which makes
the mucus more fluid. In addition H. pylori is highly motile, swimming in the
mucus and down into the crypts. Motility has been shown to be an essential
virulence factor, since non motile H. pylori has failed to infect the mucosa
in
3o experimental models Eaton et al. (Infection & Immunity 64(7}, 2445-
2448,1996).
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There are many possible reasons for this, the most obvious being an inability
to
swim down and attach to mucosal cells and the inability to avoid noxious
agents
in the stomach.
s Despite a strong apparent host immune response to H. pylori, with production
of
both local (mucosal) as well as systemic antibodies, the pathogen persists in
the
gastric mucosa, normally for the life of the host. The reason for this is
probably
that the spontaneously induced immune-responses are inadequate or directed
towards the wrong epitopes of the antigens. Alternatively the immune response
~o could be of the wrong kind, since the immune system might treat H. pylori
as a
commensal (as indicated from the life-time host/bacteria relationship).
In order to understand the pathogenesis and immunology of H. pylori
infections, it
is of great importance to define the antigenic structure of this bacterium. In
is particular, there is a need for characterization of surface-exposed,
surface
associated as well as secreted proteins which, in many bacterial pathogens,
have
been shown to constitute the main virulence factors, and which can be useful
for
the diagnosis of H. pylori and in the manufacture of vaccine compositions. If
such
proteins in addition to being surface associated also are essential for
survival
zo and/or colonization their usefulness as a target for vaccine mediated
immunotherapy targets increase.
Whenever stressed or threatened, the H. pylori cell transforms from a
bacillary to a
coccoid form. In the coccoid form, the H. pylori cell is much less sensitive
to
is antibiotics and other anti-bacterial agents. Circumstantial evidence
indicate the H.
pylori might be transmitted between indi~ iuals in ~.zis form, possibly via
water or
direct contact (oral-oral; feacal-oral). An efricient vaccine composition
should
therefore elicit an immune response towards both the coccoid and the bacillary
form of H. pylori. Since systemic immunity probably only plays a limited role
in
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protection against mucosal infections, it is also important that the vaccine
composition will enhance protective immune mechanisms locally in the stomach.
Flagellar Hook protein
Flagellar hooks from H. pylori has been shown to be composed of FIgE subunits
of
78 kDa (O'Toole et al. Molecular Microbiology,14(4), 691-703,1994). The role
of
the flagellar hook is to connect the flagella with the submembraneous
fiagellar
motor. The part of the hook extruding outside the membrane is short,
io approximately 60 nanometers (compared to approximately 10 micrometers for
the
flagella). Like the fagellum of H. pylori the hook is probably covered with a
sheet
(Geis et al. (1993) J. Med. Microbiol. 38(5), 371-377).
The amino acid sequence of the FIgE polypeptide has significant resemblance
with
is that of other known hook proteins, including limited homology to other
Helicobacter species like mustelae (O'Toole et al., supra). Polyclonal
antibodies raised
against the FIgE polypeptide showed cross-reactivity against flagellar
proteins A
and B, possibly indicating the existence of shared epitopes. Production of
FIgE
knockout H. pylori, resulted in an aflagellar, non-motile bacteria, where FlgE
2o polypeptide still was produced but could only be recovered in the
cytoplasm.
BRIEF DESCRIPTION OF THE DRAWINGS
2s Fig. 1:
Effect of therapeutic immunization of H. pylori infected mice (n=9-10/group)
with
FIgE polypeptide. Results are given as mean~SEM of number of H. pylori
associated with antnzln (=A), corpus (=B) or totally (A+C) (=C).
Abbreviations:
CFU, colony forming units (number of bacteria); unshaded bars=DOC + CT,
so Phosphate buffered saline with 0.5% deoxycholate given together with
cholera
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toxin 10 ~g/mouse; shaded bars=FIgE + CT, mice given 100 ~,g FIgE and 10 ~.g
cholera toxin. The decrease in cfu was significant in the antrum and as
calculated
for the whole stomach.
** p<0.01; * p<0.05 (Wilcoxon-Mann-Whittney sign rank test).
_. . 5
Fig. 2:
Serum IgG from mice measured by ELISA technique: response to infection and to
immunisation with FIgE. The values are expressed as mean titers ~ SEM. n=9-
10/group. ELISA coated with H. pylori strain 244: As a sign of infection H.
pylori
to specific antibodies can be found in serum in animals treated with DOC + CT
(=A.
Control/244). Following immunization with FIgE + cholera toxin (=B. FIgE/244)
this reactivity increased 4 fold (** p<0.01; Wilcoxon-Mann-Whittney sign rank
test). C=FIgE specific. Specific FIgE IgG increased in animals given FIgE +
CT, but
could not be detected in control animals.
IS
DISCLOSURE OF THE INVENTION
The purpose of this invention is to provide an antigenic H. pylori polypeptide
zo which can be useful for eliciting a protective immune response against, and
for
diagnosis of, H. pylori infection. This purpose has been achieved by the
recombinant cloning of an H. pylori gene which encodes a well conserved
essential
polypeptide. The nucleic acid sequence of this gene is similar to the sequence
of
the f 1gE gene as published by O'Toole et al., Molecular Microbiology,14(4),
691-
25 703,1994. Be"- ; an essen!-ial protein for motility, the flgE gene is
expressed by all
T ?. pylori stra~ . ~.
It has surprisingly been found that the H. pylori FIgE polypeptide, in spite
of the
facts that only a small part of the hook protein is existing outside bacteria
and that
3o it is probably covered by a sheet, can serve as a therapeutic antigen in an
H. pylori
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infected mouse model, when given together with the adjuvant cholera toxin. The
experimental data below thus indicates that the H. pylori FlgE poiypeptide,
when
used as an oral immunogen, acts as a stimulator of an immune response leading
to
a significant reduction of colonization of H. pylori in mice which were
infected
with H. pylori one month prior to immunization.
These results strongly support the use of the H. pylori FlgE polypeptide in an
oral
vaccine formulation for the use in humans to treat and prevent H. pylori
infections.
As such, the FlgE polypeptide will be useful both for the detection of H.
pylori
~o infections as well as for the manufacture of vaccine compositions, which
when
given in an appropriate pharmaceutical formulation will elicit a protective or
therapeutic immune response against such infections.
Consequently, in one aspect the present invention provides a Helicobacter
pylori
cs FIgE polypeptide for use in inducing a protective immune response to
Helicobacter
pylori infection. The term "HeIicobacter pylori FIgE polypeptide" is intended
to
mean the polypeptide which is disclosed by O'Toole et al. in Molecular
Microbiology,14(4), 691-703,1994, and which is encoded by the gene whose
nucleotide sequence is set forth as SEQ ID NO: 1, or can be obtained from the
2o National Center for Biotechnology Information (Accession number U09549), or
a
substantially similar modified form of the said polypeptide retaining
functionally
equivalent antigenicity.
The term "protective immune response" is to be understood as an immune
~s response which makes the composition suitable for therapeutic and/or
prophylactic purposes.
The term "functionally equivalent antigenicity" is to be understood as the
ability
to induce a systemic and mucosal immune response while decreasing the number
30 of H. pylori cells associated with the gastric mucosa. The skilled person
will be able
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6
to identify modified forms of the FIgE polypeptide retaining functionally
equivalent antigenicity, by use of known methods, such as epitope mapping with
in vivo induced antibodies.
In a preferred form of the invention, the Helicobacter pylori FIgE
polypeptide, for
use in inducing a protective immune response to Helicobacter pylori infection,
has
substantially the amino acid sequence set forth as SEQ ID NO: 2 in the
Sequence
Listing, or is a modified form thereof retaining functionally equivalent
antigenicity.
~o
It is thus to be understood that the definition of the Helicobacter pylori
FIgE
polypeptide is not to be limited strictly to a polypeptide with an amino acid
sequence identical with SEQ ID NO: 2 in the Sequence Listing. Rather the
invention encompasses polypeptides carrying modifications like substitutions,
is small deletions, insertions or inversions, which polypeptides nevertheless
have
substantially the biological activities of the Helicobacter pylori FIgE
polypeptide and
is retaining functionally equivalent antigenicity. Included in the definition
of the
Helicobacter pylori FIgE polypeptide are consequently polypeptides, the amino
acid
sequence of which is at least 90% homologous, preferably at least 95%
2o homologous, with the amino acid sequence set forth as SEQ ID NO: 2 in the
Sequence Listing.
In another aspect, the invention provides a vaccine composition for inducing a
protective immune response to Helicobacter pylori infection, comprising an
2s immunogenically effective amount of a Helicobacter pylori FIgE polypeptide
as
defined above, optionally together with a pharmaceutically acceptable carrier
nr
diluent.
In the present context the term "immunologically effective amount" is to be
3o understood as an amount which elicits a significant protective Helicobacter
pylori
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7
response, which will eradicate a H. pylori infection in an infected mammal or
prevent the infection in a susceptible mammal. Typically an immunologically
effective amount will comprise approximately 1 ~,g to 1000 mg, preferably
approximately 10 ~.g to 100 mg, of H. pylori antigen for oral administration,
or
approximately less than 100 ~.g for parenteral administration.
The vaccine composition comprises optionally in addition to a pharmaceutically
acceptable carrier or diluent one or more other immunologically active
antigens
for prophylactic or therapeutic use. Physiologically acceptable carriers and
~o diluents are well known to those skilled in the art and include e.g.
phosphate
buffered saline (PBS), or, in the case of oral vaccines, HC03- based
formulations or
enterically coated powder formulations.
The vaccine composition can optionally include or be administered together
with
is acid secretion inhibitors, preferably proton pump inhibitors (PPIs), e.g.
omeprazole. The vaccine can be formulated in known delivery systems such as
liposomes, ISCOMs, cochleates, etc. (see e.g. Rabinovich et al. (1994) Science
265,
1401-1404) or be attached to or incorporated into polymer microspheres of
degradable or non-degradable nature. The antigens could be associated with
live
zo attenuated bacteria, viruses or phages or with killed vectors of the same
kind. The
antigens can be chemically or genetically coupled to carrier proteins of inert
or
adjuvantic types (i.e Cholera B subunit). Consequently, the invention provides
in a
further aspect a vaccine composition according to above, in addition
comprising
an adjuvant, such as a cholera toxin. Such pharmaceutically acceptable forms
of
~s cholera toxin are known in the art, e.g. from Rappuoli et al. (1995) Int.
Arch.
Allergy & Immunol. 108(4), 327-333; and Dickinson et al. (1995) Infection and
Immunity 63(5),1617-1623.
A vaccine composition according to the invention can be used for both
therapeutic
3o and prophylactic purposes. Consequently, the invention includes a vaccine
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composition according as defined above, for use as a therapeutic or a
prophylactic
vaccine in a mammal, including man, which is infected by Helicobacter pylori.
In
this context the term "prophylactic purpose" means to induce an immune
response which will protect against future infection by Helicobacter pylori,
while
the term "therapeutic purpose" means to induce an immune response which can
eradicate an existing Helicobacter pylori infections.
The vaccine composition according to the invention is preferably administered
to
any mammalian mucosa exemplified by the buccal, the nasal, the tonsillar, the
~o gastric, the intestinal (small and large intestine), the rectal and the
vaginal mucosa.
The mucosal vaccines can be given together with for the purpose appropriate
adjuvants. The vaccine can also be given orally, or parenterally, by the
subcutaneous, intracutaneous or intramuscular route, optionally together with
the
appropriate adjuvant. The vaccine composition can optionally be given together
~s with antimicrobial therapeutic agents.
In a further aspect, the invention proivides the use of a Helicobacter pylori
FIgE
polypeptide, as defined above, in the manufacture of
(i) a composition for the treatment, prophylaxis or diagnosis of Helicobacter
pylori
2o infection;
(ii) a vaccine for use in eliciting a protective immune response against
HeIicobacter
pylori; and
(iii) a diagnostic kit for diagnosis of Helicobacter pylori infection.
2s In yet a : ,: r ther aspect, the invention provides a method of in vitro
diagnosis of
Helicobacter pylori infection comprising at least one step wherein a
Helicobacter
pylori FIgE polypeptide as defined above, optionally labelled or coupled to a
solid
support, is used. The said method could e.g. comprise the steps (a) contacting
a
said Helicobacter pylori FIgE polypeptide, optionally bound to a solid
support, with
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a body fluid taken from a mammal; and (b) detecting antibodies from the said
body fluid binding to the said FlgE polypeptide. Preferred methods of
detecting
antibodies are ELISA (Enzyme linked immunoabsorbent assay) methods which are
well known in the art.
In another aspect the invention provides a diagnostic kit for the detection of
Helicobacter pylori infection in a mammal, including man, comprising
components
which enable the method of in vitro diagnosis as described above to be carried
out.
The said diagnostic kit could e.g. comprise: (a) a Helicobacter pylori FIgE
io polypeptide; and (b) reagents for detecting antibodies binding to the said
FIgE
polypeptide. The said reagents for detecting antibodies could e.g. be an
enzyme-
labelled anti-immunoglobulin and a chromogenic substrate for the said enzyme.
In yet a further aspect, the invention provides a method of eliciting in a
mammal,
~s including humans, a protective immune response against Helicobacter pylori
infection, said method comprising the step of administering to the said mammal
an immunologically effective amount of a Helicobacter pylori FIgE polypeptide
as
defined above, or alternatively administering to the said mammal an
immunologically effective amount of a vaccine composition as defined above.
EXPERIMENTAL METHODS
Throughout this description the terms "standard protocols" and "standard
is procedures", when used in the context of molecular cloning techniques, are
to be
understood as protocols and procedures found in an ordinary laboratory manual
such as: Current Protocols in Molecular Biology, editors F. Ausubel et al.,
John
Wiley and Sons, Inc.1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T.,
Molecular
Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
so Cold Spring Harbor, NY 1989.
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Preparation of recombinant Helicobacter pylori FIgE polypeptide
DNA sequence Information
Sequence information for the gene encoding for the FIgE polypeptide was
obtained from the National Center for Biotechnology Information (Accession
number U09549; SEQ ID NO: 1).
~o PCR Amplification and cloning of DNA sequences containing ORF's for
membrane and
secreted proteins from the j99 Strain of HeIicobacter pylori.
Sequences were cloned from the J99 strain of H. pylori by amplification
cloning
using the polymerase chain reaction (PCR). Synthetic oligonucleotide primers
(see
is below) specific for the 5'- and 3'-ends of open reading frames of genes
were
designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA).
Forward primers (specific for the 5'-end of the sequence) for FlgE were
designed
to include an NcoI cloning site at the extreme 5'-terminus, while reverse
primers
included a EcoRI site at the extreme 5'-terminus to permit cloning of each H.
pylori
2o sequence into the reading frame of the pET28b vector. Inserts cloned into
the NcoI-
EcoRI sites of the pET-28b vector are fused to a vector DNA sequence encoding
an
additional 20 carboxy-terminal amino including six histidine residues (at the
extreme C-terminus).
zs Forward primer (SEQ ID NO: 3):
5'-TAT ACC ATG GTG CTT AGG TCT TTA T-3'
Reverse primer (SEQ ID I'~lU: 4):
5'-GCG AAT TCA ATT GCT TAA GAT TCA A-3'
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Genomic DNA prepared from the J99 strain of Helicobacter pylori was used as
the
source of template DNA for PCR amplification reactions (Current Protocols in
Molecular Biology, editors F. Ausubel et aL, John Wiley and Sons, Inc. 1994).
To
amplify a DNA sequence containing an H. pylori ORF, genomic DNA (50 ng) was
s introduced into a reaction vial containing 2 mM MgCl2,1 ~.M synthetic
oligonucleotide primers (forward and reverse primers) complementary to and
flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate
dATP, dGTP, dCTP, dTTP, and 2.5 units of heat stable DNA polymerase
(Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final
volume
io of 100 ~.1. The following thermal cycling conditions were used to obtain
amplified
DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System
9600 thermal cycler:
Denaturation at +94°C for 2 min;
2 cycles at +94°C for 15 sec, +30°C for 15 sec and +72°C
for 1.5 min;
~s 23 cycles at +94°C for 15 sec, +58°C for 15 sec and
+72°C for 1.5 min;
Reactions were concluded at +72°C for 6 minutes.
Upon completion of thermal cycling reactions, each sample of amplified DNA was
washed and purified using the Qiaquick Spin PCR purification kit (Qiagen,
2o Gaithersburg, MD, USA). Amplified DNA samples were subjected to digestion
with the restriction endonucleases NdeI and EcoRI according to standard
procedures. DNA samples were then subjected to electrophoresis on 1.0
NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was
visualized by exposure to ethidium bromide and long wave UV irradiation. DNA
2s contained in slices isolated from the agarose gel was purified using the
Bio 101
GeneClean Kit protocol (Bio 101 Vista, CA, USA).
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Cloning of H. pylori DNA sequences into the pET-28b prokaryotic expression
vector.
The pET-28b vector was prepared for cloning by digestion with NcoI and EcoRI
according to standard procedures. Following digestion, DNA inserts were cloned
according to standard procedures into the previously digested pET-28b
expression
vector. Products of the ligation reaction were then used to transform the BL21
strain of E. coli as described below.
Transformation of competent bacteria with recombinant plasmids
~o
Competent bacteria, E. coli strain BL21 or E. coli strain BL21(DE3), were
transformed with recombinant pET expression plasmids carrying the cloned H.
pylori sequences according to standard methods. Briefly,1 ~.l of ligation
reaction
was mixed with 50 ~.1 of electrocompetent cells and subjected to a high
voltage
is pulse, after which, samples were incubated in 0.45 ml SOC medium (0.5%
yeast
extract, 2.0% tryptone,10 mM NaCI, 2.5 mM KC1,10 mM MgCl2,10 mM MgS04
and 20, mM glucose) at +37°C with shaking for 1 hour. Samples were then
spread
on LB agar plates containing 25 ~.g/ml kanamycin sulfate for growth overnight.
Transformed colonies of BL21 were then picked and analyzed to evaluate cloned
2o inserts as described below.
Identification of recombinant pET expression plasmids carrying H. pylori
sequences
Individual BL21 clones transformed with recombinant pET-28b H. pylori genes
2s were analyzed by PCR amplification of the cloned inserts using the same
forward
and reverse primers, specific fo~ ~ ..~h H. pylori sequence, tl. ' were used
in ~. _
original PCR amplification cloning reactions. Successful amp~ification
verified the
integration of the H. pylori sequences in the expression vector according to
standard procedures.
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Isolation and Preparation of plasmid DNA from BL22 transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned H.
pylori ORFs were picked and incubated in 5 ml of LB broth plus 25 ~.g/ml
kanamycin sulfate overnight. The following day plasmid DNA was isolated and
purified using the Qiagen plasmid purification protocol (Qiagen Inc.,
Chatsworth,
CA, USA).
io Expression of recombinant H. pylori sequences in E. coli
The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174,
HB101,
JM109, DHSa, etc. for the purpose of cloning or plasmid preparation. Hosts for
expression include E. coli strains containing a chromosomal copy of the gene
for
is T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda
derivative that carries the lacl gene, the lacUV5 promoter and the gene for T7
RNA
polymerase. T7 RNA polymerase is induced by addition of isopropyl-~i-D-
thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target
plasmid, such as pET-28b, carrying its gene of interest. Strains used in our
20 laboratory include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J.,
and
Dubendorff, J.W. (1990) Methods Enzymol. 185, 60-89).
To express recombinant H. pylori sequences, 50 ng of plasmid DNA isolated as
described above was used to transform competent BL21(DE3) bacteria as
is described above (provided by Novagen as part of the pET expression system
kit).
Transformed cells were cultured in SOC medium for 1 hour, and the culture was
then plated on LB plates containing 25 ~,g/ml kanamycin sulfate. The following
day, bacterial colonies were pooled and grown in LB medium containing
kanamycin sulfate (25 ~.g/ml) to an optical density at 600 nm of 0.5 to 1.0
O.D.
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units, at which point,1 mM IPTG was added to the culture for 3 hours to induce
gene expression of the H. pylori recombinant DNA constructions .
After induction of gene expression with IPTG, bacteria were pelleted by
centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at
4°C.
Pellets were resuspended in 50 ml cold 10 mM Tris-HCI, pH 8.0, 0.1 M NaCI and
0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min
at
+4°C. Wet pellets were weighed and frozen at -80°C until ready
for protein
purification.
~o
Analytical Methods
The concentrations of purified protein preparations were quantified
spectrophotometrically using absorbance coefficients calculated from amino
acid
is content (Perkins, S.J. 1986 Eur. J. Biochem. 157,169-180). Protein
concentrations
were also measured by the method of Bradford, M.M. (1976) Anal.. Biochem. 72,
248-254, and Lowry, O.H., Rosebrough,N., Farr, A.L. & Randall, R.J. (1951) ,
using
bovine serum albumin as a standard.
2o Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels (12% or 4 to 25
gradient acrylamide) were purchased from BioRad (Hercules, CA, USA), and
stained with Coomassie Brilliant Blue. Molecular mass markers included rabbit
skeletal muscle myosin (200 kDa), E. coli ~3-galactosidase (116 kDa), rabbit
muscle
phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45
2s kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5
kDa),
egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
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Purification of FIgE from inclusion bodies
The following steps were carried out at +4°C. Cell pellets were
resuspended in
lysis buffer with 10% glycerol 200 ~.g/ml lysozyme, 5 mM EDTA,1 mM PMSF and
s 0.1% (3-mercaptoethanol. After passage through the cell disrupter, the
resulting
homogenate was made 0.2% DOC, stirred 10 minutes, then centrifuged (10,000 g x
30 min). The pellets were first washed with lysis buffer containing 10%
glycerol,10
mM EDTA, 1% Triton X-100,1 mM PMSF and 0.1% (3-mercaptoethanol, then with
lysis buffer containing 1 M urea, l mM PMSF and 0.1% ~i-mercaptoethanol. The
~o resulting white pellet was composed primarily of inclusion bodies, free of
unbroken cells and membranous materials.
The following steps were carried out at room temperature. Inclusion bodies
were
dissolved in 20 ml 8 M urea in lysis buffer with 1 mM PMSF and 0.1% ~3-
is mercaptoethanol, and incubated at room temperature for 1 hour. Materials
that
did not dissolve were removed by centrifugation (100,000 x g for 30 min) . The
clear supernatant was filtered and loaded onto a Ni2+-NTA agarose column
equilibrated in 8 M urea in lysis buffer. The column was washed with 250 ml
(50
bed volumes) of lysis buffer containing 8 M urea, l mM PMSF and 0.1% ~i-
2o mercaptoethanol, and developed with sequential steps of lysis buffer
containing 8
M urea, 1 mM PMSF, 0.1% (3-mercaptoethanol and 20,100, 200, and 500 mM
imidazole. Fractions were monitored by absorbance at OD28p nm, and peak
fractions were analyzed by SDS-PAGE. Two bands were visualized by Coomassie
Brilliant Blue staining, a major band Mr = 78 kDa and a minor band Mr = 60
kDa.
2s Purity of recombinant FIgE (7$ kDa) was assessed at greater than 90%. As
with the
purification of the soluble proteins, fractions containing the recombinant
protein
eluted at 100 mM imidazole.
Urea was slowly removed from the FIgE polypeptide by dialysis against TBS
3o containing 0.5% DOC with sequential reduction in urea as follows; 6M, 4M,
3M,
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16
2M,1M, 0.5 M then 0 M. Each dialysis step was carried for a minimum of 4 hours
at room temperature,
After dialysis, samples were concentrated by pressure filtration using Amicon
s stirred cells. Protein concentrations were then measured by the methods of
Perkins, Bradford and Lowry.
EXAMPLES OF THE INVENTION
EXAMPLE 1: THERAPEUTIC IMMUNIZATION
1. Materials f~ Methods
is 1.1 Animals
Female SPF BALB/c mice were purchased from Bomholt Breeding centre
(Denmark). They were kept in ordinary makrolon cages with free supply of water
and food. The animals were 4-6 weeks old at arrival.
1.2. Infection
After a minimum of one week of acclimatization, the animals were infected with
a
type 2 strain of H. pylori (strain 244, originally isolated from an ulcer
patient). This
2s strain has earlier proven to be a good colonizer of the mouse stomach.
Bacteria
from a stock kept at -70°C were gro«~n overnight in Brucella broth
supplemented
with 10% fetal calf serum, at +37°C L~ a microaerophilic atmosphere
(10% CO2, 5%
02). The animals were given an oral dose of omeprazole (400 ~,mol/kg) and
after
3-5 h an oral inoculation of H. pylori (approximately 10~-10$ CFU/animal).
3o Infection was checked in control animals 2-3 weeks after the inoculation.
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1.3. Immunizations
One month after infection, two groups of mice (10 mice/group) were immunized 4
times over a 34 day period (day 1,15, 25 and 35). Purified recombinant FIgE
dissolved in PBS plus 0.5% Deoxycholate (DOC) was given at a dose of 100
microgram/mouse.
As an adjuvant, the animals in both the control as well as the FlgE group were
also
given IO ~.g/mouse of cholera toxin (CT) with each immunization. Omeprazole
io (400 ~mol/kg) was given orally to all animals 3-5 h prior to immunization
as a
way of protecting the antigens from acid degradation. Animals were sacrificed
1-2
weeks after final immunization.
Group I: 300 ~1 PBS with 0.5% DOC containing 10 ~.g CT
Group 2: 300 ~,l PBS with 0.5% DOC containing 100 ~.g FIgE and 10 ~.g CT.
is
1.4. Analysis of infection
The mice were sacrificed by C02 and cervical dislocation. The abdomen and
chest
cavity was opened and blood sampled by heart puncture. Subsequently the
2o stomach was removed. After cutting the stomach along the greater curvature,
it
was rinsed in saline and subsequently cut into two identical pieces. An area
of 25
mm2 of the mucosa from the antrum and corpus was scraped separately with a
surgical scalpel. The mucosa scraping was suspended in Brucella broth, diluted
and plated onto Blood Skirrow plates. The plates were incubated under
2s microaerophilic conditions for 3-5 days and the number of colonies was
counted.
The identity of H. pylori was ascertained by urease and catalase test and by
direct
microscopy or Gram staining.
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18
1.5. Antibody measurements
Serum antibodies were collected from blood. Prior to centrifugation, the blood
was
diluted with equal amount of PBS. The serum was kept at -20°C until
analysis.
Serum antibodies were measured using an ELISA where plates were coated either
with a particulate fraction of H. pylori strain 244 or with FIgE followed by
addition
of different dilutions of serum. The ELISA was developed with alkaline
phosphatase-labelled anti-mouse-Ig-antibodies. The anti-Ig antibodies were of
an
anti-heavy/anti-light chain type, which should detect all types of antibodies.
io
2. Results
2.1. Therapeutic immunization: effects on CFU
is The animals in this study were infected with H. pylori strain 244 one month
prior
to immunizations. Mice in groups of ten were then immunized with either
cholera
toxin (CT) or CT together with the recombinant FIgE polypeptide. Four weeks
after the final immunization, the animals were sacrificed and CFU was
determined
(Fig. 1). The animals treated with CT alone, were highly infected both in
corpus
2o and antrum. Animals actively immunized with recombinant FIgE polypeptide
and
CT had significantly decreased CFU values in the antrum and in the stomach as
a
whole compared with the CT treated animals (p<0.01 and p<0.05, respectively;
Wilcoxon-Mann-Whittney sign rank test).
is 2.2. Therapeutic immunization: effects on antibody formation and secretion
As a sign of infection H. pylori specific antibodies can be found in serum
(Control/244). In animals given FIgE + CT the titer against strain 244 (as
membrane proteins) increased 4-fold (p<0.01). Only in animals given FIgE + CT
so could a specific serum IgG titer against FIgE be measured (Fig. 2).
CA 02293293 1999-12-10
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19
FIgE specific IgG increased in animals given FIgE + CT, but could not be
detected
in control animals.
The results presented show that the recombinant FIgE H. pylori polypeptide is
highly immunogenic when given orally, together with cholera toxin as an
adjuvant, measured as an increase in systemic FIgE specific Ig antibodies. The
immunization with FIgE also resulted in a significant increase in the Ig
titers
against a particulate fraction of H. pylori. In addition, a dramatic decrease
in
io number of colonizing H. pylori in the gastric mucosa of the infected mice
was
found following immunization with FIgE toghether with cholera toxin.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Astra AB
(B) STREET: Vastra Malarehamnen 9
(C) CITY: Sodertalje
(E) COUNTRY: Sweden
(F) POSTAL CODE (ZIP): S-151 85
(G) TELEPHONE: +46 8 553 260 00
(H) TELEFAX: +46 8 553 288 20
(ii) TITLE OF INVENTION: Vaccine Compositions V
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2550 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:321..2477
(D) OTHER INFORMATION:/product= "FlgE flagellar hook
protein"
(x) PUBLICATION INFORMATION:
(A) AUTHORS: O'Toole, Paul W.
Kostrzynska, Magdalena
Trust, Trevor J.
(B) TITLE: Non-motile mutants of Helicobacter pylori and
Helicobacter mustelae defective in flagellar hook
production
(C) JOURNAL: Mol. Microbiol.
(D) VOLUME: 14
(E) ISSUE: 4
(F) PAGES: 691-703
(G) DATE: 1994
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AACAAAGCGA TAACTCCTTT GTCTTATTAG CGACACAATT TAACCCATTG ACTTTAAATC 60
GCGCTTCAGC CGAAGAGATT CAAGATCATG AATGCGCGAT TTTGCACTAA AGCGAGTTAG 120
ATTCTTAAAT TTGAGCGATA ACCTTTAAAA AGCGTAATTA AGGGGTGGTG TTACAAAACC 180
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21
CCCTATCCCC TTATGAATTT GACCGATCTT TTTGATTAAC AAAACTTTAA AATCCGCAAT 240
CAATCATTCT AAAAAGCTAT TTAGGAACAA CTTTTGCTTT ATTTTGCATA GATTGAATTT 300
CTTTAAATTA AAGGATAACC 350
ATG CTT AGG
TCT TTA TGG
TCT GGT GTC
AAT
Met sn
Leu
Arg
Ser
Leu
Trp
Ser
Gly
Val
A
1 5 10
GGG ATGCAA GCCCACCAA ATCGCTTTGGAT ATTGAGAGT AACAATATT 398
Giy MetGln AlaHisGln IleAlaLeuAsp IleGluSer AsnAsnIle
15 20 25
GCG AACGTG AATACCACT GGTTTTAAGTAT TCTAGGGCT TCTTTTGTG 446
Ala AsnVal AsnThrThr GlyPheLysTyr SerArgAla SerPheVal
30 35 40
GAT ATGCTT TCTCAAGTC AAACTCATCGCT ACCGCACCC TATAAAAAC 494
Asp MetLeu SerGlnVal LysLeuIleAla ThrAlaPro TyrLysAsn
45 50 55
GGG TTAGCA GGGCAGAAT GATTTTTCTGTG GGGCTTGGG GTAGGCGTG 542
Gly LeuAla GlyGlnAsn AspPheSerVal GlyLeuGly ValGlyVal
60 65 70
GAT GCGACG ACTAAAATC TTTTCACAAGGC AATATCCAA AACACAGAT 590
Asp AlaThr ThrLysIle PheSerGlnGly AsnIleGln AsnThrAsp
75 80 85 90
GTC AAAACC GATCTAGCG ATTCAAGGCGAT GGCTTTTTT ATCATTAAC 638
Val LysThr AspLeuAla IleGlnGlyAsp GlyPhePhe IleIleAsn
95 100 105
CCT GATAGG GGGATCACG CGCAATTTCACT AGAGATGGG GAGTTCCTT 686
Pro AspArg GlyIleThr ArgAsnPheThr ArgAspGly GluPheLeu
110 115 120
TTT GACTCG CAAGGGAGT TTGGTTACCACC GGCGGGCTT GTGGTGCAA 734
Phe AspSer GlnGlySer LeuValThrThr GlyGlyLeu ValValGln
125 130 135
GGG TGGGTG AGAAATGGG AGCGATACCGGC AATAAAGGG AGCGATACA 782
Gly TrpVal ArgAsnGly SerAspThrGly AsnLysGly SerAspThr
140 145 150
GAC GCTTTA AAAGTGGAT AACACCGGTCCT TTAGAAAAC ATTAGGATT 830
Asp AlaLeu LysValAsp AsnThrGlyPro LeuGluAsn IleArgIle
155 160 165 170
GAT CCTGGA ATGGTGATG CCAGCCAGAGCG AGTAACCGC ATTTCTATG 878
Asp ProGly MetValMet ProAlaArgAla SerAsnArg IleSerMet
175 180 185
AGG GCGAAT TTAAACGCT GGAAGGCATGCC GATCAAACA GCGGCGATA 926
Arg AlaAsn LeuAsnAla GlyArgHisAla AspGlnThr AlaAlaIle
190 195 200
TTC GCTTTG GATTCTTCA GCCAAAACCCCT TCAGATGGC ATTAATCCG 974
Phe AlaLeu AspSerSer AlaLysThrPro SerAspGly IleAsnPro
205 210 215
GTG TATGAT TCAGGCACG AATCTTGCTCAA GTCGCCGAA GACATGGGA 1022
Val TyrAsp SerGlyThr AsnLeuAlaGln ValAlaGlu AspMetGly
220 225 230
TCT TTATAC AATGAAGAT GGCGACGCTCTT TTGTTGAAT GAAAATCAA 1070
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22
SerLeuTyr AsnGluAsp GlyAspAlaLeu LeuLeuAsn GluAsnGln
235 240 245 250
GGGATTTGG GTGAGCTAT AAGAGTCCAAAA ATGGTCAAA GACATCCTC 1118
GlyIleTrp ValSerTyr LysSerProLys MetValLys AspIleLeu
255 260 265
CCTTCTGCA GAAAACAGC ACGCTTGAATTG AATGGCGTT AAGATTTCT 1166
ProSerAla GluAsnSer ThrLeuGluLeu AsnGlyVal LysIleSer
270 275 280
TTCACAAAC GATTCAGCG GTGAGCCGGACT TCAAGCTTA GTGGCGGCT 1214
PheThrAsn AspSerAla ValSerArgThr SerSerLeu ValAlaAla
285 290 295
AAAAATGCG ATCAATGCA GTCAAAAGCCAA ACAGGCATT GAAGCTTAT 1262
LysAsnAla IleAsnAla ValLysSerGln ThrGlyIle GluAlaTyr
300 305 310
TTAGACGGC AAGCAATTG CGTTTGGAAAAC ACCAATGAA TTAGACGGC 1310
LeuAspGly LysGlnLeu ArgLeuGluAsn ThrAsnGlu LeuAspGly
315 320 325 330
GATGAAAAG CTTAAAAAC ATTGTAGTTACT CAAGCCGGA ACCGGAGCG 1358
AspGluLys LeuLysAsn IleValValThr GlnAlaGly ThrGlyAla
335 340 345
TTCGCTAAC TTTTTAGAC GGCGATAAAGAT GTAACGGCT TTCAAATAC 1406
PheAlaAsn PheLeuAsp GlyAspLysAsp ValThrAla PheLysTyr
350 355 360
AGCTACACG CATTCTATT AGCCCTAACGCC AATAGCGGG CAGTTTAGG 1454
SerTyrThr HisSerIle SerProAsnAla AsnSerGly GlnPheArg
365 370 375
ACCACTGAA GACTTGCGC GCCTTAATCCAG CATGACGCT AATATCGTT 1502
ThrThrGlu AspLeuArg AlaLeuIleGln HisAspAla AsnIleVal
380 385 390
AAAGATCCT AGCCTAGCG GACAATTACCAA GACTCAGCC GCTTCTATA 1550
LysAspPro SerLeuAla AspAsnTyrGln AspSerAla AlaSerIle
395 400 405 410
GGAGTTACA ATCAACCAA TACGGCATGTTT GAAATCAAC AATAAAGAC 1598
GlyValThr IleAsnGln TyrGlyMetPhe GluIleAsn AsnLysAsp
415 420 425
AATAAAAAT GTCATTAAA GAAAATCTTAAT ATCTTTGTG AGCGGGTAT 1646
AsnLysAsn ValIleLys GluAsnLeuAsn IlePheVal SerGlyTyr
430 435 440
TCTTCAGAC AGCGTAACG AACAATGTTTTG TTTAAAAAT GCGATGAAA 1694
SerSerAsp SerValThr AsnAsnValLeu PheLysAsn AlaMetLys
445 450 455
GGGCTTAAT ACCGCTTCT TTAATTGAAG-'9GGAGCGTCA GCGAGCAGT 1742
GlyLeuAsn ThrAlaSer LeuIleGlur GlyA.aSer AlaSerSer
,~
460 465 470
TCTAAATTC ACCCACGCT ACGCATGCGACA AGCATTGAT GTGATAGAC 1790
SerLysPhe ThrHisAla ThrHisAlaThr SerIleAsp ValIleAsp
475 480 485 490
AGCTTAGGC ACTAAACAC GCCATGCGCATT GAGTTTTAT AGGAGTGGG 1838
SerLeuGly ThrLysHis AlaMetArgIle GluPheTyr ArgSerGly
495 500 505
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23
GGA GCG GAT TGG AAT TTT AGA GTG ATC GTG CCT GAG CCT GGG GAA TTA 1886
Gly Ala Asp Trp Asn Phe Arg Val Ile Val Pro Glu Pro Gly Glu Leu
510 515 520
GTA GGG GGG TCA GCG GCT AGG CCT AAT GTG TTT GAA GGA GGC CGT TTG 1934
Val Gly Gly Ser Ala Ala Arg Pro Asn Val Phe Glu Gly Gly Arg Leu
- 525 530 535
CAC TTCAATAATGAC GGATCGCTTGCA GGCATG CCGCCTCTTTTG 1982
AAC
His PheAsnAsnAsp GlySerLeuAla GlyMetAsn ProProLeuLeu
540 545 550
CAA TTTGACCCTAAA AATGGTGCTGAT GCCCCCCAA CGCATCAATTTA 2030
Gln PheAspProLys AsnGlyAlaAsp AlaProGln ArgIleAsnLeu
555 560 565 570
GCT TTTGGTTCCTCA GGGAGTTTTGAC GGGCTAACG AGCGTGGATAAG 2078
Ala PheGlySerSer GlySerPheAsp GlyLeuThr SerValAspLys
575 580 585
ATT TCTGAAACTTAT GCGATTGAGCAA AACGGCTAT CAAGCGGGCGAT 2126
Ile SerGluThrTyr AlaIleGluGln AsnGlyTyr GlnAlaGlyAsp
590 595 600
TTG ATGGATGTCCGC TTTGATTCAGAT GGGGTGCTT TTAGGAGCGTTC 2174
Leu MetAspValArg PheAspSerAsp GlyValLeu LeuGlyAlaPhe
605 610 615
AGT AATGGCAGGACT TTAGCGCTCGCT CAAGTGGCT TTAGCGAATTTC 2222
Ser AsnGlyArgThr LeuAlaLeuAla GlnValAla LeuAlaAsnPhe
620 625 630
GCT AACGATGCGGGC TTGCAGGCTTTA GGCGGGAAT GTCTTTTCTCAA 2270
Ala AsnAspAlaGly LeuGlnAlaLeu GlyGlyAsn ValPheSerGln
635 640 645 650
ACC GGAAACTCAGGG CAAGCCTTAATC GGTGCGGCT AATACGGGGCGT 2318
Thr GlyAsnSerGly GlnAlaLeuIle GlyAlaAla AsnThrGlyArg
655 660 665
AGG GGTTCAATTTCA GGATCTAAACTG GAGTCTAGT AATGTGGATTTG 2366
Arg GlySerIleSer GlySerLysLeu GluSerSer AsnValAspLeu
670 675 680
AGC CGGAGTTTAACG AATTTGATTGTG GTTCAAAGG GGCTTTCAAGCA 2414
Ser ArgSerLeuThr AsnLeuIleVal ValGlnArg GlyPheGlnAla
685 690 695
AAC TCTAAAGCGGTA ACCACATCCGAT CAAATCCTT AATACCCTATTG 2462
Asn SerLysAlaVal ThrThrSerAsp GlnIleLeu AsnThrLeuLeu
700 705 710
AAT CTTAAGCAATAA ACTAAAGGAT TACTCTAATA 2517
CAATATAATA
GGGGCTAATT
Asn LeuLysGln
715
TAAAGATTAA GGTTTAGTAT GCATGAATAC TCG 2550
. (2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 719 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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24
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Arg Ser Leu Trp Ser Gly Val Asn Gly Met Gln Ala His Gln
1 5 10 15
Ile Ala Leu Asp Ile Glu Ser Asn Asn Ile Ala Asn Val Asn Thr Thr
20 25 30
Gly Phe Lys Tyr Ser Arg Ala Ser Phe Val Asp Met Leu Ser Gln Val
35 40 45
Lys Leu Ile Ala Thr Ala Pro Tyr Lys Asn Gly Leu Ala Gly Gln Asn
50 55 60
Asp Phe Ser Val Gly Leu Gly Val Gly Val Asp Ala Thr Thr Lys Ile
65 70 75 80
Phe Ser Gln Gly Asn Ile Gln Asn Thr Asp Val Lys Thr Asp Leu Ala
85 90 95
Ile Gln Gly Asp Gly Phe Phe Ile Ile Asn Pro Asp Arg Gly Ile Thr
100 105 110
Arg Asn Phe Thr Arg Asp Gly Glu Phe Leu Phe Asp Ser Gln Gly Ser
115 120 125
Leu Val Thr Thr Gly Gly Leu Val Val Gln Gly Trp Val Arg Asn Gly
130 135 140
Ser Asp Thr Gly Asn Lys Gly Ser Asp Thr Asp Ala Leu Lys Val Asp
145 150 155 160
Asn Thr Gly Pro Leu Glu Asn Ile Arg Ile Asp Pro Gly Met Val Met
165 170 175
Pro Ala Arg Ala Ser Asn Arg Ile Ser Met Arg Ala Asn Leu Asn Ala
180 185 190
Gly Arg His Ala Asp Gln Thr Ala Ala Ile Phe Ala Leu Asp Ser Ser
195 200 205
Ala Lys Thr Pro Ser Asp GIy Ile Asn Pro Val Tyr Asp Ser Gly Thr
210 215 220
Asn Leu Ala Gln Val Ala Glu Asp Met Gly Ser Leu Tyr Asn Glu Asp
225 230 235 240
Gly Asp Ala Leu Leu Leu Asn Glu Asn Gln Gly Ile Trp Val Ser Tyr
245 250 255
Lys Ser Pro Lys Met Val Lys Asp Ile Leu Pro Ser Ala Glu Asn Ser
260 265 270
Thr Leu Glu L~PU Asn Gly Val Lys Ile Ser Phe Thr Asn Asp Ser Ala
275 280 285
Val Ser Arg Thr Ser Ser Leu Val Ala Ala Lys Asn Ala Ile Asn AIa
290 295 300
Val Lys Ser Gln Thr Gly Ile Glu Ala Tyr Leu Asp Gly Lys Gln Leu
305 310 315 320
Arg Leu Glu Asn Thr Asn Glu Leu Asp Gly Asp Glu Lys Leu Lys Asn
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325 330 335
Ile Val Val Thr Gln Ala Gly Thr Gly Ala Phe Ala Asn Phe Leu Asp
340 345 350
Gly Asp Lys Asp Val Thr Ala Phe Lys Tyr Ser Tyr Thr His Ser Ile
355 360 365
Ser Pro Asn Ala Asn Ser Gly Gln Phe Arg Thr Thr Glu Asp Leu Arg
370 375 380
Ala Leu Ile Gln His Asp Ala Asn Ile Val Lys Asp Pro Ser Leu Ala
385 390 395 400
Asp Asn Tyr Gln Asp Ser Ala Ala Ser Ile Gly Val Thr Ile Asn Gln
405 410 415
Tyr Gly Met Phe Glu Ile Asn Asn Lys Asp Asn Lys Asn Val Ile Lys
420 425 430
Glu Asn Leu Asn Ile Phe Val Ser Gly Tyr Ser Ser Asp Ser Val Thr
435 440 445
Asn Asn Val Leu Phe Lys Asn Ala Met Lys Gly Leu Asn Thr Ala Ser
450 455 460
Leu Ile Glu Gly Gly Ala Ser Ala Ser Ser Ser Lys Phe Thr His Ala
465 470 475 480
Thr His Ala Thr Ser Ile Asp Val Ile Asp Ser Leu Gly Thr Lys His
485 490 495
Ala Met Arg Ile Glu Phe Tyr Arg Ser Gly Gly Ala Asp Trp Asn Phe
500 505 510
Arg Val Ile Val Pro Glu Pro Gly Glu Leu Val Gly Gly Ser Ala Ala
515 520 525
Arg Pro Asn Val Phe Glu Gly Gly Arg Leu His Phe Asn Asn Asp Gly
530 535 540
Ser Leu Ala Gly Met Asn Pro Pro Leu Leu Gln Phe Asp Pro Lys Asn
545 550 555 560
Gly Ala Asp Ala Pro Gln Arg Ile Asn Leu Ala Phe Gly Ser Ser Gly
565 570 575
Ser Phe Asp Gly Leu Thr Ser Val Asp Lys Ile Ser Glu Thr Tyr Ala
580 585 590
Ile Glu Gln Asn Gly Tyr Gln Ala Gly Asp Leu Met Asp Val Arg Phe
595 600 605
Asp Ser Asp Gly Val Leu Leu Gly Ala Phe Ser Asn Gly Arg Thr Leu
610 615 620
Ala Leu Ala Gln Val Ala Leu Ala Asn Phe Ala Asn Asp Ala Gly Leu
625 630 635 640
Gln Ala Leu Gly Gly Asn Val Phe Ser Gln Thr Gly Asn Ser Gly Gln
645 650 655
Ala Leu Ile Gly Ala Ala Asn Thr Gly Arg Arg Gly Ser Ile Ser Gly
660 665 670
Ser Lys Leu Glu Ser Ser Asn Val Asp Leu Ser Arg Ser Leu Thr Asn
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26
675 680 685
Leu Ile Val Val Gln Arg GIy Phe Gln Ala Asn Ser Lys Ala Val Thr
690 695 700
Thr Ser Asp Gln Ile Leu Asn Thr Leu Leu Asn Leu Lys Gln
705 710 715
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TATACCATGG TGCTTAGGTC TTTAT 25
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GCGAATTCAA TTGCTTAAGA TTCAA 25
