Note: Descriptions are shown in the official language in which they were submitted.
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RAINBOW TROUT IL13; ENCODING NUCLEIC
ACID; MATERIALS AND METHODS
The present invention relates to polypeptides with
the function of Rainbow Trout Interleukin (IL)-10 and
has arisen from cloning of an encoding gene sequence.
The polypeptides and encoding nucleic acid are useful
in particular for disease control. Polypeptides may be
produced by expression from the encoding nucleic acid.
To date only a single interleukin sequence has
been isolated unambiguously in any non-mammalian
animal. The plethora of cytokine bioactivities in
rainbow trout, together with cross-reactivity of
several mammalian cytokines to fish leucocytes,
suggests that cytokines do have a role in fish immune
responses (Secombes et al., 1996). One of these cross-
reacting cytokines is interleukin (IL) 1. That
mammalian IL1 can cross-react to fish cells has been
known for over a decade (Hamby et al., 1986), yet
despite several groups looking for IL1 in several fish
species (Ellsaesser & Clem, 1994; Verburg van Kemenade
et al., 1995), to date no sequence data exist.
Similarly, in other vertebrate groups, especially
chickens where relatively large numbers of researchers
have been involved, no sequence data exist. One of the
reasons for the lack of success is almost certainly
that the homology of mammalian cytokines to cytokines
in other vertebrate groups is low, minimising the
chances of success. For this reason homology cloning
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was abandoned in the search for chicken interferon,
where screening of an expression library for biological
activity was the only successful approach (Sekellick et
al., 1994).
In the present study an homology cloning approach
was used to search for fish IL1. Initial studies were
carried out using degenerate forward and reverse
primers designed against regions of homology in the
mammalian sequences. Since this approach was
unsuccessful in our laboratory for IL1, as well as a
large number of other cytokines looked for including
IL2, IL6, IL8, TNFa and IFNy, an alternative homology
cloning approach was taken in further studies where
non-degenerate forward and reverse primers to the IL1
sequences were used for both ILla and ILlfi gene
sequences. This approach lead to the isolation of
trout IL1$, but in an entirely unexpected fashion.
The experimental work leading to the present
invention is described below.
Chance binding of a primer ("R1IL1Q") to two
locations of the trout cDNA probed allowed the trout
IL1Q molecule coding sequence to be isolated and
sequenced. This molecule, e.g. produced as a
recombinant, has many applications, especially for
disease control in aquaculture. Currently there is a
large interest in the use of cytokines as adjuvants for
vaccines, and IL1 is one cytokine showing promise in
mammalian studies (Lin et al., 1995; Blecha et al.,
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1995). In addition, it is clear that cytokines such as
IL1 can be used therapeutically to combat disease
(Vogels et al., 1995; BacaEstrada et al., 1995), and
that even at very low doses they can synergise with
conventional antibiotic therapy to augment survival
(Nakamura et al., 1989).
Thus, the new trout IL1Q molecule may be
administered as an adjuvant in existing and developing
fish vaccines. Since the most efficient route of
immunisation is currently intraperitoneal injection
(Tatner, 1993), as seen with vaccines for furunculosis
and Hitra disease, the IL1 preparation may be simply
mixed with the vaccine solution for injection.
Immersion vaccines also exist, where fish are bathed in
the vaccine solution. An ILl preparation may be simply
added to the bath. For therapeutic use, again many
antibiotics are given by injection and an ILl
preparation may be simply added to the existing
antibiotic preparation just prior to injection.
Antibiotics are also given in the feed, and this is
possible for trout IL1.
Encoding nucleic acid may be administered to
deliver the polypeptide to cells within a fish. The
administered nucleic acid may include a regulatory
sequence for expression of the polypeptide.
The homologies obtained (see below), together with
Northern blot analysis showing the molecule is induced
post-exposure to LPS, clearly show this molecule is
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biologically relevant to fish immune responses to Gram
negative bacteria and is equivalent to mammalian ILlf3.
According to one aspect of the present invention
there is provided a nucleic acid molecule which has a
nucleotide sequence encoding a polypeptide which
comprises the amino acid sequence shown in Figure 3
(also shown in Figure 4, bottom line).
The coding sequence may be that shown in Figure 3
or it may be a mutant, variant, derivative, allele or
homologue of the sequence shown. The sequence may
differ from that shown by a change which is one or more
of addition, insertion, deletion and substitution of
one or more nucleotides of the sequence shown. Changes
to a nucleotide sequence may result in an amino acid
change at the protein level, or not, as determined by
the genetic code.
Thus, nucleic acid according to the present
invention may comprise a sequence different from the
sequence shown in Figure 3, yet encode a polypeptide
with the same amino acid sequence. The amino acid
sequence shown in Figure 3 consists of 260 residues.
On the other hand the encoded polypeptide may
comprise an amino acid sequence which differs by one or
more amino acid residues from the amino acid sequence
shown in Figure 3. Nucleic acid encoding a polypeptide
which is an amino acid sequence variant, derivative,
allele, mutant or homologue of the sequence shown in
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Figure 3 is further provided by the present invention.
Such polypeptides are discussed below. Nucleic acid
encoding such a polypeptide may show greater than about
60% homology with the coding sequence shown in Figure
5 3, greater than about 70% homology, greater than about
80% homology, greater than about 90% homology or
greater than about 9511 homology.
Generally, nucleic acid according to the present
invention is provided as an isolate, in isolated and/or
purified form, or free or substantially free of
material with which it is naturally associated, such as
free or substantially free of nucleic acid flanking the
IL1 gene in the trout genome, except possibly one or
more regulatory sequence(s) for expression. Nucleic
acid may be wholly or partially synthetic and may be
genomic DNA, cDNA or RNA.
Nucleic acid may be provided as part of a
replicable vector, and also provided by the present
invention are a vector comprising nucleic acid as set
out above, particularly any expression vector from
which the encoded polypeptide can be expressed under
appropriate conditions, and a host cell containing any
such vector or nucleic acid. An expression vector in
this context is a nucleic acid molecule comprising
nucleic acid encoding a polypeptide of interest and
appropriate regulatory sequences for expression of the
polypeptide, either in an in vitro expression system,
e.g. reticulocyte lysate, or in vivo, e.g. in
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eukaryotic cells such as COS or CHO cells or in prokaryotic
cells such as E. coli. This is discussed further below.
The present invention also provides a cell containing
nucleic acid or a vector as according to the invention, which
cell is comprised in a fish, and a fish including such a cell.
Nucleic acid according to the present invention includes
nucleic acid encoding a polypeptide which comprises the amino
acid sequence shown in Figure 3 (also Figure 4, bottom line),
of an allele, mutant, variant or derivative thereof, which
nucleic acid comprises the coding nucleotide sequence shown on
the first page of Figure 3, or an allele, mutant, variant or
derivative thereof. The nucleotide sequence shown on the first
page of Figure 3 may be used to obtain the full-length trout
coding sequence by a variety of methods. For instance,
oligonucleotide probes or primers may be designed to match
fragments of the sequence, particularly fragments of relatively
rare sequence, based on codon usage or statistical analysis.
Nucleic acid according to the present invention is
obtainable using one or more oligonucleotides or
polynucleotides, designed on the basis of sequence information
provided herein, as probes or primers. For instance, the
nucleotide sequence provided on the first page of Figure 3 may
be used as a probe. Nucleic acid isolated and/or purified from
one or more cells of trout or other fish, or a nucleic acid
library derived from nucleic acid isolated and/or purified from
the fish (e.g. a cDNA library derived from mRNA isolated from
the fish), may be probed under conditions for
AMENDED SHEET
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selective hybridisation and/or subjected to a specific
nucleic acid amplification reaction such as the
polymerase chain reaction (PCR). The nucleic acid
probed or used as template in the amplification
reaction may be genomic DNA, cDNA or RNA. if
necessary, one or more gene fragments may be ligated to
generate a full-length coding sequence. This is
discussed further below.
A further aspect of the present invention provides
a polypeptide which has the amino acid sequence shown
in Figure 3 (also shown in Figure 4, bottom line),
which may be in isolated and/or purified form, free or
substantially free of material with which it is
naturally associated, such as trout polypeptides other
than IL10, or (for example if produced by expression in
a prokaryotic cell) lacking in native glycosylation,
e.g. unglycosylated.
Polypeptides which are amino acid sequence
variants, alleles, derivatives, mutants and homologues
are also provided by the present invention. A
polypeptide which is a variant, allele, derivative,
mutant or homologue may have an amino acid sequence
which differs from that given in Figure 3 by one or
more of addition, substitution, deletion and insertion
of one or more amino acids. Preferred such
polypeptides have IL1Q function, that is to say have
one or more of the following properties: immunological
cross-reactivity with an antibody reactive with one or
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more of sheep, cervus, bovine, horse, pig, mouse,
human, rat, macmu, certo, rabbit and felca IL1,6;
sharing an epitope with the trout IL1(3 for which the
amino acid sequence is shown in Figure 3 (as determined
for example by immunological cross-reactivity between
the polypeptide in question and the native trout IL10;
one or more cytokine activities, e.g. such as to
characterise the polypeptide as an interleukin,
preferably IL1, for instance ability to stimulate
leucocytes in a mitogen-induced proliferation assay,
and/or the ability to increase the expression of IL2
receptors on T-cells, and/or ability to enhance
antigen-specific acivity of T-helper cells, and/or the
ability to stimulate production of prostaglandins
and/or any other metabolite of arachidonic acid; a
biological activity which is inhibited by an antibody
raised against an IL113, particularly an antibody raised
against the polypeptide whose sequence is shown in
Figure 3; adjuvant effect, for instance on
administration to a fish such as a trout; synergism
with antibiotic therapy to counter bacterial infection.
A polypeptide which is an amino acid sequence
variant, allele, derivative, mutant or homologue of the
amino acid sequence shown in Figure 3 may comprise an
amino acid sequence which shares greater than about 35%
sequence identity with the sequence shown, greater than
about 40%, greater than about 50%, greater than about
60%, greater than about 70%, greater than about 80%,
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greater than about 90% or greater than abou 95%. The
sequence may share greater than about 60% similarity,
greater than about 70% similarity, greater than about
80% similarity or greater than about 90% similarity
with the amino acid sequence shown in Figure 3.
Particular amino acid sequence variants may differ from
that shown in Figure 3 by insertion, addition,
substitution or deletion of 1 amino acid, 2, 3, 4, 5-
10, 10-20 20-30, 30-50, 50-100, 100-150, or more than
150 amino acids.
The sequence information provided herein and the
results of comparison with known mammalian IL].
sequences shows that the trout IL10 amino acid sequence
shows approximately 30% identity (49-57% similarity)
with the known mammalian sequences.
A convenient way of producing a polypeptide
according to the present invention is to express
nucleic acid encoding it, by use of the nucleic acid in
an expression system.
Accordingly, the present invention also
encompasses a method of making a polypeptide (as
disclosed), the method comprising expression from
nucleic acid according to the present invention
encoding the polypeptide. This may conveniently be
achieved by growing a host cell in culture, containing
such a vector, under appropriate conditions which cause
or allow expression of the polypeptide. Polypeptides
may also be expressed in in vitro systems, such as
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reticulocyte lysate.
Systems for cloning and expression of a
polypeptide in a variety of different host cells are
well known. Suitable host cells include bacteria,
5 eukaryotic cells such as mammalian and yeast, and
baculovirus systems. Mammalian cell lines available in
the art for expression of a heterologous polypeptide
include Chinese hamster ovary cells, HeLa cells, baby
hamster kidney cells, COS cells and many others. A
10 common, preferred bacterial host is E. coli.
Suitable vectors can be chosen or constructed,
containing appropriate regulatory sequences, including
promoter sequences, terminator fragments,
polyadenylation sequences, enhancer sequences, marker
genes and other sequences as appropriate. Vectors may
be plasmids, viral e.g. 'phage, or phagemid, as
appropriate. For further details see, for example,
Molecular Cloning: a Laboratory Manual: 2nd edition,
Sambrook et al., 1989, Cold Spring Harbor Laboratory
Press. Many known techniques and protocols for
manipulation of nucleic acid, for example in
preparation of nucleic acid constructs, mutagenesis,
sequencing, introduction of DNA into cells and gene
expression, and analysis of proteins, are described in
detail in Short Protocols in Molecular Biology, Second
Edition, Ausubel et al. eds., John Wiley & Sons, 1992.
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The provision of the novel trout ILl/3 enables for
the first time the production of antibodies able to
bind it specifically. Accordingly, a further aspect of
the present invention provides an antibody able to bind
specifically to the polypeptide whose sequence is given
in Figure 3. Such an antibody may be specific for trout
ILlfl or for fish ILlQ's in the sense of being able to
distinguish between the polypeptide it is able to bind
and the other bovine, cerco, cervus, feline, horse,
human, macmu, mouse, ovine, rabbit, rat and scrofa
IL1f3's for which it has no or substantially no binding
affinity (e.g. a binding affinity of 1000x worse than
for ILIO). Trout ILlp-specific antibodies bind an
epitope on the trout molecule which is either not
present or is not accessible on the other IL1R
molecules. Antibodies according to the invention may
be specific for trout IL1,6 as between that molecule and
other trout antigens, so as to be useful in
purification of the trout IL1f3 and potentially other
fish ILl3's, which may facilitate their cloning from
expression libraries.
Preferred antibodies according to the invention
are isolated, in the sense of being free from
contaminants such as antibodies able to bind other
polypeptides and/or free of serum components.
Monoclonal antibodies are preferred for some purposes,
though polyclonal antibodies are within the scope of
the present invention.
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Antibodies may be obtained using techniques which
are standard in the art. Methods of producing
antibodies include immunising a mammal (eg mouse, rat,
rabbit, horse, goat, sheep or monkey) with the protein
or a fragment thereof. Antibodies may be obtained from
immunised animals using any of a variety of techniques
known in the art, and screened, preferably using
binding of antibody to antigen of interest. For
instance, Western blotting techniques or
immunoprecipitation may be used (Armitage et al, 1992,
Nature 357: 80-82).
As an alternative or supplement to immunising a
mammal with a peptide, an antibody specific for a
protein may be obtained from a recombinantly produced
library of expressed immunoglobulin variable domains,
eg using lambda bacteriophage or filamentous
bacteriophage which display functional immunoglobulin
binding domains on their surfaces; for instance see
W092/01047. The library may be naive, that is
constructed from sequences obtained from an organism
which has not been immunised with any of the proteins
(or fragments), or may be one constructed using
sequences obtained from an organism which has been
exposed to the antigen of interest.
Antibodies according to the present invention may
be modified in a number of ways. Indeed the term
"antibody" should be construed as covering any binding
substance having a binding domain with the required
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specificity. Thus the invention covers antibody
fragments, derivatives, functional equivalents and
homologues of antibodies, including synthetic molecules
and molecules whose shape mimicks that of an antibody
enabling it to bind an antigen or epitope.
Example antibody fragments, capable of binding an
antigen or other binding partner are the Fab fragment
consisting of the VL, VH1 Cl and CH1 domains; the Fd
fragment consisting of the VH and CH1 domains; the Fv
fragment consisting of the VL and VH domains of a
single arm of an antibody; the dAb fragment which
consists of a VH domain; isolated CDR regions and
F(ab')2 fragments, a bivalent fragment comprising two
Fab fragments linked by a disulphide bridge at the
hinge region. Single chain Fv fragments are also
included.
A hybridoma producing a monoclonal antibody
according to the present invention may be subject to
genetic mutation or other changes. It will further be
understood by those skilled in the art that a
monoclonal antibody can be subjected to the techniques
of recombinant DNA technology to produce other
antibodies or chimeric molecules which retain the
specificity of the original antibody. Such techniques
may involve introducing DNA encoding the immunoglobulin
variable region, or the complementarity determining
regions (CDRs), of an antibody to the constant regions,
or constant regions plus framework regions, of a
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different immunoglobulin. See, for instance,
EP184187A, GB 2188638A or EP-A-0239400. Cloning and
expression of chimeric antibodies are described in EP-
A-0120694 and EP-A-0125023.
Hybridomas capable of producing antibody with
desired binding characteristics are within the scope of
the present invention, as are host cells, eukaryotic or
prokaryotic, containing nucleic acid encoding
antibodies (including antibody fragments) and capable
of their expression. The invention also provides
methods of production of the antibodies comprising
growing a cell capable of producing the antibody under
conditions in which the antibody is produced, and
preferably secreted.
Antibodies according to the present invention may
be used in screening for the presence of a polypeptide,
for example in a test sample comprising cells or cell
lysate, and may be used in purifying and/or isolating a
polypeptide according to the present invention, for
instance following production of the polypeptide by
expression from encoding nucleic acid therefor.
After production by expression from encoding
nucleic acid, a polypeptide according to the present
invention may be isolated and/or purified (e.g. using
an antibody as discussed). The isolated and/or
purified polypeptide may then be used in formulation of
a composition, which may comprise at least one
additional component, for example a pharmaceutical
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composition comprising a pharmaceutically acceptable
excipient, vehicle or carrier.
A composition comprising a polypeptide according
to the invention may be used in prophylactic and/or
5 therapeutic treatment, particularly of fish. For
instance, the polypeptide may be used as an adjuvant
for immunisation, e.g. by injection or bathing of fish.
For therapeutic use, the polypeptide may be
administered with an antibiotic, for instance as a
10 combined preparation for simultaneous or sequential
administration, to capitalise on the synergism between
IL1 and antibiotics which has been demonstrated.
Administration is preferably in a
"prophylactically effective amount" or a
15 "therapeutically effective amount" (as the case may
be), this being sufficient to show benefit to an
individual. The actual amount administered, and rate
and time-course of administration, will depend on the
nature and severity of what is being treated.
Prescription of treatment of fish, eg decisions on
dosage etc, is within the responsibility of
veterinarians. Administration may be alone or in
combination with other treatments, either
simultaneously or sequentially dependent upon the
condition to be treated.
Pharmaceutical compositions for administration in
accordance with the present invention may comprise a
pharmaceutically acceptable excipient, carrier, buffer,
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stabiliser or other materials well known to those
skilled in the art. Such materials should be non-toxic
and should not interfere with the efficacy of the
active ingredient. The precise nature of the carrier
or other material will depend on the route of
administration, the most efficient of which is
currently intraperitoneal injection.
For injection, the active ingredient will be in
the form of an acceptable solution which is pyrogen-
free and has suitable pH, isotonicity and stability.
Those of relevant skill in the art are well able to
prepare suitable solutions. Preservatives, stabilisers,
buffers, antioxidants and/or other additives may be
included, as required.
Further aspects of the present invention provide a
method of making a pharmaceutical composition
comprising formulation by admixture of a polypeptide as
disclosed with a pharmaceutically acceptable excipient,
vehicle or carrier, and use of a polypeptide as
disclosed in the manufacture of a medicament,
particularly for administration to fish in order to
capitalise on a property of the polypeptide (as
discussed above).
A further aspect of the present invention provides
an oligonucleotide or polynucleotide fragment of the
nucleotide sequence shown in Figure 3, or a
complementary sequence, for use in a method of
obtaining nucleic acid encoding a polypeptide with ILlp
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function from other fish. One such polynucleotide
fragment is shown on the first page of Figure 3. Such
a method may comprise hybridisation of two probes or
primers to target nucleic acid. The hybridisation may
be as part of a PCR procedure, or as part of a probing
procedure not involving PCR. An example procedure
would be a combination of PCR and low stringency
hybridisation. A screening procedure, chosen from the
many available to those skilled in the art, is used to
identify successful hybridisation events and isolated
hybridised nucleic acid.
The sequence provided in Figure 3 is itself useful
for identifying nucleic acid encoding other I11/3
proteins. Accordingly, the present invention provides
a method of obtaining nucleic acid encoding a protein
with ILl0 activity, the method comprising hybridisation
of a probe having the sequence shown in Figure 3 or a
complementary sequence, to target nucleic acid.
Hybridisation is generally followed by identification
of successful hybridisation and isolation of nucleic
acid which has hybridised to the probe. The method may
involve one or more steps of PCR (see below). It will
not always be necessary to use a probe with the
complete sequence shown in Figure 3. Shorter
fragments, particularly fragments with a sequence
conserved between the trout IL10 and one or more of the
mammalian sequences, may be used. The fragment whose
sequence is provided on the first page of Figure 3 may
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be used. Nucleic acid which has some alteration, e.g.
addition, insertion, deletion or substitution of one or
more nucleotides, in the sequence will be useful,
provided the degree of homology with one of the
sequence given is sufficiently high.
Where the nucleic acid is double-stranded DNA,
hybridisation will generally be preceded by denaturing
to produce single-stranded DNA. Probing may employ the
standard Southern blotting technique. For instance DNA
may be extracted from cells of a fish of interest and
digested with different restriction enzymes.
Restriction fragments may then be separated by
electrophoresis on an agarose gel, before denaturation
and transfer to a nitrocellulose filter. Labelled
probe may be hybridised to the DNA fragments on the
filter and binding determined. DNA for probing may be
prepared from RNA preparations from cells of a fish of
interest.
Binding of a probe to target nucleic acid (e.g.
DNA) may be measured using any of a variety of
techniques at the disposal of those skilled in the art.
For instance, probes may be radioactively,
fluorescently or enzymatically labelled. Other methods
not employing labelling of probe include examination of
restriction fragment length polymorphisms,
amplification using PCR, RNAase cleavage and allele
specific oligonucleotide probing.
Preliminary experiments may be performed by
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hybridising under low stringency conditions various
probes to Southern blots of DNA digested with
restriction enzymes. Suitable conditions would be
achieved when a large number of hybridising fragments
were obtained while the background hybridisation was
low. Using these conditions cDNA libraries
representative of expressed sequences in the fish of
interest may be searched for an IL1/3-encoding sequence.
Where a full-length encoding nucleic acid molecule
has not been obtained, a smaller molecule representing
part of the full molecule, may be used to obtain full-
length clones. Inserts may be prepared from partial
cDNA clones and used to screen cDNA libraries. The
full-length clones isolated may be subcloned into
expression vectors and IL10 activity assayed by
transfection into suitable host cells, e.g. with a
reporter plasmid.
As an alternative to probing, though still
employing nucleic acid hybridisation, oligonucleotides
designed to amplify DNA sequences may be used in PCR
reactions or other methods involving amplification of
nucleic acid, using routine procedures. See for
instance "PCR protocols; A Guide to Methods and
Applications", Eds. Innis et al, 1990, Academic Press,
New York.
Preferred amino acid sequences suitable for use in
the design of probes or PCR primers are sequences
conserved (completely, substantially or partly) between
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at least two IL10 polypeptides, e.g. with any of the
amino acid sequences shown in Figure 4.
On the basis of amino acid sequence information,
oligonucleotide probes or primers may be designed,
5 taking into account the degeneracy of the genetic code,
and, where appropriate, codon usage of the organism
from the candidate nucleic acid is derived. An
oligonucleotide for use in nucleic acid amplification
may have about 10 or fewer codons (e.g. 6, 7 or 8),
10 i.e. be about 30 or fewer nucleotides in length (e.g.
18, 21 or 24). Those skilled in the art are well
versed in the design of primers for use in PCR.
Assessment of whether or not such a PCR product
corresponds to an ILl0 gene may be conducted in various
15 ways. A PCR band from such a reaction might contain a
complex mix of products. Individual products may be
cloned and each one individually screened, for example
by determination of IL1# function, or binding of a
specific antibody or binding fragment thereof, upon
20 expression in a suitable expression system.
Aspects of the present invention will now be
illustrated with reference to the accompanying figures,
by way of example and not limitation. Further aspects
and embodiments will be apparent to those of ordinary
skill in the art.
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Figure 1 shows (Figure 1(a)) ILla human (topline)
and consensus (asterisks) nucleotide sequences, and
(Figure 1(b)) IL10 human (topline) and consensus
(asterisks) nucleotide sequences. Coding sequence
alignments were made across all known species for each
cytokine using the Genetics Computer Group Sequence
Analysis Software Package, Version 7. Nucleotide
consensus across species is denoted by the presence of
a "*" symbol below the human cytokine sequence. The
narrow line represents XZAP vector, while the 5'
untranslated region is indicated upstream of the IL10
coding sequence.
Figure 2 shows the position of primers used to
probe for the trout ILlf3 sequence.
Figure 3 shows the Rainbow Trout interleukin 1
(IL1)$ nucleotide sequence obtained as described
herein, including some 5' untranslated sequence, the
IL1/3 coding sequence and the encoded amino acid
sequence. In bold: ATG = Start codon for translation
(M = methionine); TAA = Stop codon for translation;
NYS, NTS and NLS = glycosylation sites; AATAAA =
polyadenylation signal; ATTTA = RNA stability motif for
cytokines. The predicted translation of the trout IL1fl
consists of 260 amino acids, with approximately 30%
identity (49-57% similarity) with the known mammalian
IL10 amino acid sequences. The nucleotide sequence has
55% identity in a 550 bp overlap with mammalian
sequences, particularly at the 3' end of the translated
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peptide where most conservation is seen between-
mammals.
Figure 4 shows multiple alignments of the trout
IL1/3 amino acid sequence of Figure 3 with other known
IL1R sequences. Note the weak overall conservation,
with one or two conserved motifs towards the 5' end.
Figure 5 shows an unrooted phylogenetic tree
showing the relationship between the predicted trout
IL1Q amino acid sequence and other known IL1(3
sequences. The tree was constructed by the "neighbour
joining" method using the CLUSTAL W and PHYLIP
packages, and was bootstrapped 10,000 times.
MATERIALS AND METHODS
Library construction
Since cytokine genes are not constitutively
expressed, in vivo stimulation with an attenuated
bacterial pathogen was used in an attempt to switch on
the relevant fish cytokine genes. Two days after
bacterial challenge head kidney macrophages were
partially purified and mRNA extracted for library
construction using a Stratagene ZAP cDNATMsynthesis kit.
This library was subsequently used in a PCR based
approach to search for fish cytokine genes.
Primers
Primers were designed against conserved regions of
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the known mammalian IL1 sequences (Figure 1). These
included two forward and two reverse primers for IL1a
and one forward and reverse primer for Ilfl. Further
primers were designed against the initial trout ILlf3
sequence obtained to allow cloning of the full length
molecule.
ILla
Forward (F) 1: tagtgaattcgaagaatacagttc
Forward (F) 2: acgggaattctctgaagaagagacgg
Reverse (R) 1: gattgaattccaaccgtctcttcttc
Reverse (R) 2: catggaattcccagaagaagaggag
ILl
Forward (F) 1: cagccatggcaaccgtacctg
Forward (F) 2: atcagtaccctggagtctgc
Forward (F) 3: ggatcagctgaagtccatc
Forward (F) 4: cgaattcatggattttgagtca
Reverse (R) 1: ttgagaagtgctgatgtacc
Reverse (R) 2: cagcttggagctccatgctg
Reverse (R) 3: cttagttgtggcgctggatg
PCR, cloning and sequencing
The above primers were used in various
combinations with cDNA from the constructed macrophage
gene library. Obtained products were cloned using a TA
cloning kit (Invitrogen), plasmid DNA prepared from
bacterial colonies and sequenced on an ABI automated
CA 02269085 1999-04-14
WO 98/17802 PCT/GB97/02855
24
373A sequencer. The nucleotide and amino acid sequence
data were then compared with entries on the GenBank
data base. Subsequently anchored PCR was used to
obtain the 5' and 3' ends of the sequence using the
universal primers T3 and T7 in combination with new
trout IL1,6 specific primers F2, F3 and R2 (see Figure 2
for primer positions), giving products of approximately
250 bp with T3/R2 and 650 bp with F2/T7. The full
length clone was obtained using F4 and R, and was
approximately 780 bp.
RT-PCR of cDNA from a variety of tissues (brain,
pituitary, gill, liver, blood leucocytes, spleen and
kidney macrophages) was used to look for sites of ILlf3
expression in rainbow trout.
Northern blots
The full length clone had one EcoRl cut site, and
was cut to give a 550 bp probe for Northern analysis.
The probe was labelled with 32P and used for
hybridisation to RNA from control and LPS stimulated
head kidney macrophages to see if IL1p expression was
inducible by an inflammatory insult.
Results
All combinations of ILla primers failed to give
products with homology to IL1. The IL10 primers gave a
faint band of approximately 750 bp, that was
reamplified and cloned. When sequenced this product
CA 02269085 1999-04-14
WO 98/17802 PCT/GB97/02855
was found to be homologous to mammalian ILl$. However,
analysis of the primer sites revealed that the F1 ILlg
primer had not annealed to the fish sequence, and that
instead the Ri primer had annealed twice, once at the
5 predicted site but also in a complementary fashion to a
region just upstream of the start codon. Thus, the
product was obtained very much as a chance event.
The 5' end was obtained by anchored PCR using
universal primer T3 with trout specific primer R2, and
10 the 3' end was obtained by anchored PCR using a nested
strategy with F3 and T7 followed by F2 and T7.
Finally, several full length clones were obtained using
primers F4 and R3 and a consensus sequence of 4 clones
obtained (Figure 3).
15 The nucleotide sequence was translated and both
sequences were compared with entries in Genbank. As
seen in Tables 1 and 2, all the top "hits" were for
ILlf3 and the ILlf3 receptor antagonist (a related
molecule), with between 49-57% similarity to mammalian
20 IL10. The translation was also used to construct an
alignment of the trout sequence with mammalian
sequences (Figure 4) and to construct a phylogenetic
tree (Figure 5), where clear relatedness to the known
sequences was apparent, but far less than anticipated.
25 RT-PCR with a variety of tissues revealed that
expression only occurred in lymphoid sites (blood
leucocytes, kidney macrophages and spleen). In
addition, Northern blot analysis showed that whilst a
CA 02269085 1999-04-14
WO 98/17802 PCT/GB97/02855
26
low level expression of IL10 was detectable in kidney
macrophages, expression was increased following
stimulation with LPS.
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27
REFERENCES
1. BacaEstrada et al., (1995) Interferon Cytokine
Res. 15: 431-439.
2. Blecha et al., (1995) Vet. Immunol. Immunopathol.
44: 329-346.
3. Ellsaesser & Clem (1994) Cytokine 6: 10-20.
4. Hamby et al., (1986) Lymphokine Res. 5: 157-162
5. Lin et al., (1995) Clin. Infect. Dis. 21: 1439-
1449.
6. Nakamura (1989) Antimicrob. Agents Chemother. 33:
1804.
7. Secombes et al., (1996) Fish Shellfish Immunol. 6:
291-304.
8. Sekellick et al., (1994) J. Interferon Res. 14:
71-79.
9. Tatner (1993) Fish Vaccines In: Vaccines for
veterinary applications (ed by Peters, A.R.)
Butterworth-Heinemann Ltd, Oxford pp 199-224.
10. Verburg van Kemanade et al., (1995) Dev. Comp.
Immunol. 19: 59-70.
11. Vogels et al., (1995) Antimicrob. Agents
Chemother. 39: 1744-1747.
CA 02269085 1999-04-14
WO 98117802 PCT/GB97/02855
28
Table I. Best scores in the swissprot database between the obtained trout
sequence and
all other known sequences.
Code Description init I initn opt
illb-pig interleukin-1 beta precursor 99 176 225
illb-bovine interleukin-1 beta precursor 114 164 227
illb-sheep interleukin-1 beta precursor 112 162 220
illb-felca interleukin-I beta precursor 93 161 198
illb-certo interleukin-1 beta precursor 100 149 231
illb-macmu interleukin-1 beta precursor 100 149 235
iilb-human interleukin-1 beta precursor 99 148 238
ilib-mouse interleukin-I beta precursor 107 144 239
illb-rabbit interleukin-1 beta precursor 105 141 228
illx-mouse inierleuldn-1 receptor antagonist 84 112 167
illx-rat interleukin-I receptor antagonist 84 104 164
ehol-human epilepsy holoprosencephaly 42 96 53
it lx-rabit int:erleukin-1 receptor antagonist 74 90 158
illx-human interleukin-1 receptor antagonist 73 88 166
After the top 14 "Bits" the scores fall below 40, 85 and 40 respectively,
suggesting the homology
is so low that it is insignificant.
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29
Table 2. Percentage similarity of the full length trout ILI (3 amino acid
sequence with other
known ILI Q sequences.
Species Homology
sheep 57.1%
cervus 55.7%
bovine 55.3%
horse 54.5%
pig 54.0%
mouse 53.8%
human 52.5%
rat 52.1%
macmu 52.1%
certo 51.6%
rabbit 51.5%
felca 49.0%
CA 02269085 1999-10-18
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: The University Court of the University of
Aberdeen
(B) STREET: Regent Walk
(C) CITY: Aberdeen
(E) COUNTRY: United Kingdom
(F) POSTAL CODE (ZIP): AB24 3FX
(ii) TITLE OF INVENTION: Rainbow trout ILl-beta; Encoding nucleic
acid; Materials and methods
(iii) NUMBER OF SEQUENCES: 27
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bereskin & Parr
(B) STREET: Suite 4000, Box 401, 40 King Street West
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): M5H 3Y2
(v) COMPUTER READABLE FORM:
(A) COMPUTER: IBM PC compatible
(B) OPERATING SYSTEM: PC-DOS/MS-DOS
(C) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,269,085
(B) FILING DATE: 16-OCT-1997
(C) CLASSIFICATION: C12N 15/25, C07K 14/545, A61K 38/20,
C12N 5/10, A01K 67/027, A61K 48/00
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9621681.7
(B) FILING DATE: 17-OCT-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9623173.3
(B) FILING DATE: 06-NOV-1996
(viii) PATENT AGENT INFORMATION:
(A) NAME: Gravelle, Micheline
(B) REGISTRATION NUMBER: 4189
(C) REFERENCE NUMBER: 420-288
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1330 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 85..864
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CA 02269085 1999-10-18
31
CACAAGAACT AAGGACTGAA TACAAGACAA CTGCTACCTA CCTACAAACT TCAAAACAAC 60
TCATTAAGAT CTAACTACAC AGAC ATG GAT TTT GAG TCA AAC TAC AGT TTA 111
Met Asp Phe Glu Ser Asn Tyr Ser Leu
1 5
ATA AAG AAC ACC TCT GAA AGT GCA GCA TGG AGC TCC AAG CTG CCT CAG 159
Ile Lys Asn Thr Ser Glu Ser Ala Ala Trp Ser Ser Lys Leu Pro Gin
15 20 25
GGT CTG GAT CTG GAG GTA TCC CAT CAC CCC ATC ACC ATG CGT CAC ATT 207
Gly Leu Asp Leu Glu Val Ser His His Pro Ile Thr Met Arg His Ile
30 35 40
GCC AAC CTC ATC ATC GCC ATG GAG AGG TTA AAG GGT GGC GAG GGG GTT 255
Ala Asn Leu Ile Ile Ala Met Glu Arg Leu Lys Gly Gly Glu Gly Val
45 50 55
ACC ATG GGA ACC GAG TTC AAG GAC AAG GAC CTG CTC AAC TTC TTG CTG 303
Thr Met Gly Thr Glu Phe Lys Asp Lys Asp Leu Leu Asn Phe Leu Leu
60 65 70
GAG AGT GCT GTG GAA GAA CAT ATA GTG TTG GAG TTG GAG TCG GCA CCC 351
Glu Ser Ala Val Glu Glu His Ile Val Leu Glu Leu Glu Ser Ala Pro
75 80 85
CCA GCG AGC AGG AGG GCA GCA GGG TTC AGC AGT ACA TCA CAG TAT GAG 399
Pro Ala Ser Arg Arg Ala Ala Gly Phe Ser Ser Thr Ser Gin Tyr Glu
90 95 100 105
TGT AGC GTC ACT GAC TCT GAG AAC AAG TGC TGG GTC CTG ATG AAT GAG 447
Cys Ser Val Thr Asp Ser Glu Asn Lys Cys Trp Val Leu Met Asn Glu
110 115 120
GCT ATG GAG CTG CAC GCC ATG ATG CTC CAG GGA GGC AGC AGC TAC CAC 495
Ala Met Glu Leu His Ala Met Met Leu Gin Gly Gly Ser Ser Tyr His
125 130 135
AAA GTG CAT TTG AAC CTG TCT TCG TAC GTC ACG CCC GTC CCC ATT GAG 543
Lys Val His Leu Asn Leu Ser Ser Tyr Val Thr Pro Val Pro Ile Glu
140 145 150
ACT GAA GCC AGA CCT GTA GCC CTA GGC ATA AAG GGA TCC AAC CTC TAC 591
Thr Glu Ala Arg Pro Val Ala Leu Gly Ile Lys Gly Ser Asn Leu Tyr
155 160 165
CTG TCC TGC TCC AAA TCG GGA GGC AGG CCC ACC CTG CAC CTA GAG GAG 639
Leu Ser Cys Ser Lys Ser Gly Gly Arg Pro Thr Leu His Leu Glu Glu
170 175 180 185
GTT GCG GAC AAG GAT CAG CTG AAG TCC ATC AGC CAG CAG AGC GAC ATG 687
Val Ala Asp Lys Asp Gin Leu Lys Ser Ile Ser Gin Gin Ser Asp Met
190 195 200
GTG CGT TTC CTT TTC TAC AGA CGG AAC ACC GGG GTT GAC ATC AGT ACC 735
Val Arg Phe Leu Phe Tyr Arg Arg Asn Thr Gly Val Asp Ile Ser Thr
205 210 215
CTG GAG TCT GCC AGT TTC AGG AAC TGG TTC ATC AGC ACG GAC ATG CAG 783
Leu Glu Ser Ala Ser Phe Arg Asn Trp Phe Ile Ser Thr Asp Met Gin
220 225 230
CAG GAC TAC ACC AAA CCG GTA GAC ATG TGT CAG AAG GCA GCC CCC AAC 831
Gin Asp Tyr Thr Lys Pro Val Asp Met Cys Gin Lys Ala Ala Pro Asn
235 240 245
CGC CTT ACC ACC TTC ACC ATC CAG CGC CAC AAC TAAGATGGCC GCAAACACTG 884
CA 02269085 1999-10-18
32
Arg Leu Thr Thr Phe Thr Ile Gln Arg His Asn
250 255 260
ACACAACACC AGTCAGGAGG CCAAGGCCTC CAATAGAGAT CCTCTACAAT TCATGTGATT 944
CTATTGGGCC TCTACGATCA GGACTGGGTT GCCACTGTGG GTGCACGCAC ATCAACAGAT 1004
TTAAGTGTAT TGCTTGTAAG ACACAAGAAT TTCATGAGGT TTAGAGTATG TATCGAGTAC 1064
AGCATGTTAC CATTGGGGTG TATTGACGTA GTCAATATTG TGGTAAGCGC CCCTGATATA 1124
ATCAAAATAT GCTTAAAACG AAAACTAAGT CACTATTGTT CTACTATTTA TTAATATATC 1184
TATAATTCAG TAATTTATAC TTATTTATCA ATGTATTTAT TTGGCTCTGT TGATGTTGCA 1244
CATATCCATT GATACATGCA AACATTTAAA ATTCTTATGT GCTCTTTTTA ATAAACATAT 1304
TTAAATGGAA AAAAAAAAAA AAAAAA 1330
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Asp Phe Glu Ser Asn Tyr Ser Leu Ile Lys Asn Thr Ser Glu Ser
1 5 10 15
Ala Ala Trp Ser Ser Lys Leu Pro Gln Gly Leu Asp Leu Glu Val Ser
20 25 30
His His Pro Ile Thr Met Arg His Ile Ala Asn Leu Ile Ile Ala Met
35 40 45
Glu Arg Leu Lys Gly Gly Glu Gly Val Thr Met Gly Thr Glu Phe Lys
50 55 60
Asp Lys Asp Leu Leu Asn Phe Leu Leu Glu Ser Ala Val Glu Glu His
65 70 75 80
Ile Val Leu Glu Leu Glu Ser Ala Pro Pro Ala Ser Arg Arg Ala Ala
85 90 95
Gly Phe Ser Ser Thr Ser Gln Tyr Glu Cys Ser Val Thr Asp Ser Glu
100 105 110
Asn Lys Cys Trp Val Leu Met Asn Glu Ala Met Glu Leu His Ala Met
115 120 125
Met Leu Gln Gly Gly Ser Ser Tyr His Lys Val His Leu Asn Leu Ser
130 135 140
Ser Tyr Val Thr Pro Val Pro Ile Glu Thr Glu Ala Arg Pro Val Ala
145 150 155 160
Leu Gly Ile Lys Gly Ser Asn Leu Tyr Leu Ser Cys Ser Lys Ser Gly
165 170 175
Gly Arg Pro Thr Leu His Leu Glu Glu Val Ala Asp Lys Asp Gln Leu
180 185 190
CA 02269085 1999-10-18
33
Lys Ser Ile Ser Gln Gln Ser Asp Met Val Arg Phe Leu Phe Tyr Arg
195 200 205
Arg Asn Thr Gly Val Asp Ile Ser Thr Leu Glu Ser Ala Ser Phe Arg
210 215 220
Asn Trp Phe Ile Ser Thr Asp Met Gln Gln Asp Tyr Thr Lys Pro Val
225 230 235 240
Asp Met Cys Gln Lys Ala Ala Pro Asn Arg Leu Thr Thr Phe Thr Ile
245 250 255
Gln Arg His Asn
260
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ala Thr Val Pro Glu Pro Ile Asn Glu Met Met Ala Tyr Tyr Ser
1 5 10 15
Asp Glu Asn Glu Leu Leu Phe Glu Ala Asp Asp Pro Lys Gln Met Lys
20 25 30
Ser Cys Ile Gln His Leu Asp Leu Gly Ser Met Gly Asp Gly Asn Ile
35 40 45
Gln Leu Gln Ile Ser His Gln Phe Tyr Asn Lys Ser Phe Arg Gln Val
50 55 60
Val Ser Val Ile Val Ala Met Glu Lys Leu Arg Asn Ser Ala Tyr Ala
65 70 75 80
His Val Phe His Asp Asp Asp Leu Arg Ser Ile Leu Ser Phe Ile Phe
85 90 95
Glu Glu Glu Pro Val Ile Phe Glu Thr Ser Ser Asp Glu Phe Leu Cys
100 105 110
Asp Ala Pro Val Gln Ser Ile Lys Cys Lys Leu Gln Asp Arg Glu Gln
115 120 125
Lys Ser Leu Val Leu Ala Ser Pro Cys Val Leu Lys Ala Leu His Leu
130 135 140
Leu Ser Gln Glu Met Asn Arg Glu Val Val Phe Cys Met Ser Phe Val
145 150 155 160
Gln Gly Glu Glu Arg Asp Asn Lys Ile Pro Val Ala Leu Gly Ile Lys
165 170 175
Asp Lys Asn Leu Tyr Leu Ser Cys Val Lys Lys Gly Asp Thr Pro Thr
180 185 190
Leu Gln Leu Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met
195 200 205
Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu
210 215 220
CA 02269085 1999-10-18
34
Phe Glu Ser Val Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile
225 230 235 240
Glu Glu Arg Pro Val Phe Leu Gly His Phe Arg Ala Gly Gln Asp Ile
245 250 255
Thr Asp Phe Arg Met Glu Thr Leu Ser Pro
260 265
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser
1 5 10 15
Ser Asn Glu Asp Asp Leu Phe Phe Glu Val Asp Gly Pro Lys Gln Met
20 25 30
Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile
35 40 45
Gln Leu Gln Ile Ser His Glu His Tyr Asn Lys Gly Phe Arg Gln Ala
50 55 60
Val Ser Val Val Val Ala Met Glu Lys Leu Arg Lys Met Leu Val Pro
65 70 75 80
Cys Pro Gln Thr Phe Gln Asp Asn Asp Leu Ser Thr Leu Ile Pro Phe
85 90 95
Ile Phe Glu Glu Glu Pro Val Phe Leu Asp Thr Cys Asn Asn Asp Ala
100 105 110
Cys Val His Asp Ala Pro Val Arg Ser Leu His Cys Thr Leu Arg Asp
115 120 125
Ala Gln Leu Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala
130 135 140
Leu His Leu Gln Gly Gin Asp Val Glu Gln Gln Val Val Phe Ser Met
145 150 155 160
Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Val Pro Val Ala Leu
165 170 175
Gly Leu Lys Ala Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp
180 185 190
Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys
195 200 205
Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Giu Ile Asn Asn
210 215 220
Lys Leu Glu Cys Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr
225 230 235 240
Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Arg Gly Gly
245 250 255
CA 02269085 1999-10-18
Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser
260 265
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ala Pro Val Pro Glu Pro Ile Asn Glu Met Met Ala Tyr Tyr Ser
1 5 10 15
Asp Glu Asn Glu Leu Leu Phe Glu Ala Asp Gly Pro Lys Gln Met Lys
20 25 30
Ser Cys Ile Gln His Leu Asp Leu Gly Ser Val Glu Val Gly Asn Ile
35 40 45
Gln Leu Gln Ile Ser His Gln Leu Tyr Asn Lys Ser Phe Arg Gln Val
50 55 60
Val Ser Val Ile Val Ala Met Glu Lys Leu Arg Asn Ser Ala Tyr Ala
65 70 75 80
His Val Phe His Asp Asp Asp Leu Arg Asn Val Leu Ser Phe Ile Phe
85 90 95
Glu Glu Glu Pro Val Ile Phe Glu Thr Ser Ser Asp Glu Phe Leu Cys
100 105 110
Asp Ala Ala Val Gln Ser Val Asn Cys Lys Leu Gln Asp Arg Glu Gln
115 120 125
Asn Ser Leu Val Leu Ala Ser Pro Cys Val Leu Lys Ala Leu His Leu
130 135 140
Leu Ser Gln Glu Met Ser Arg Glu Val Val Phe Cys Met Ser Phe Val
145 150 155 160
Gln Ala Glu Glu Arg Asp Asn Lys Ile Pro Val Ala Leu Gly Ile Arg
165 170 175
Asp Lys Asn Gln Tyr Leu Ser Cys Val Lys Lys Gly Asp Thr Pro Thr
180 185 190
Leu Gln Leu Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met
195 200 205
Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asp Thr Val Glu
210 215 220
Phe Glu Ser Val Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser His Pro
225 230 235 240
Glu Glu Lys Pro Val Phe Leu Gly His Phe Arg Gly Gly Gln Asp Ile
245 250 255
Thr Asp Phe Arg Met Glu Thr Leu Ser Pro
260 265
(2) INFORMATION FOR SEQ ID NO:6:
CA 02269085 1999-10-18
36
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ala Pro Val Pro Glu Leu Thr Ser Glu Met Met Ala Tyr Tyr Ser
1 5 10 15
Asp Glu Asn Asp Leu Phe Phe Glu Ala Asp Gly Pro Glu Lys Met Lys
20 25 30
Gly Ser Leu Gln Asn Leu Ser His Ser Phe Leu Gly Asp Glu Gly Ile
35 40 45
Gln Leu Gln Ile Ser His Gln Pro Asp Asn Lys Ser Leu Arg His Ala
50 55 60
Val Ser Val Ile Val Ala Met Glu Lys Leu Lys Lys Ile Ser Phe Ala
65 70 75 80
Cys Ser Gln Pro Leu Gln Asp Glu Asp Leu Lys Ser Leu Phe Cys Cys
85 90 95
Ile Phe Glu Glu Glu Pro Ile Ile Cys Asp Thr Trp Asp Asp Gly Phe
100 105 110
Val Cys Asp Ala Ala Ile Gln Ser Gln Asp Tyr Thr Phe Arg Asp Ile
115 120 125
Ser Gln Lys Ser Leu Val Leu Ser Gly Ser Tyr Glu Leu Arg Ala Leu
130 135 140
His Leu Asn Gly Gln Asn Met Asn Gln Gln Val Val Phe Arg Met Ser
145 150 155 160
Phe Val His Gly Glu Glu Asn Ser Lys Lys Ile Pro Val Val Leu Cys
165 170 175
Ile Lys Lys Asn Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Gly Lys
180 185 190
Pro Thr Leu Gln Leu Glu Met Leu Asp Pro Lys Val Tyr Pro Lys Lys
195 200 205
Lys Met Glu Lys Arg Phe Val Phe Asn Lys Thr Glu Ile Lys Gly Asn
210 215 220
Val Glu Phe Glu Ser Ser Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser
225 230 235 240
Gln Ala Glu Glu Met Pro Val Phe Leu Gly Asn Thr Lys Gly Gly Gln
245 250 255
Asp Ile Thr Asp Phe Ile Met Glu Ser Ala Ser
260 265
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02269085 1999-10-18
37
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Ala Thr Val Pro Glu Pro Ala Lys Glu Val Met Ala Asn Asn Gly
1 5 10 15
Asp Asn Asn Asn Asp Leu Leu Phe Glu Ala Asp Gly Pro Lys Glu Met
20 25 30
Lys Cys Arg Thr Gln Asn Leu Asp Leu Ser Pro Leu Gly Asp Gly Ser
35 40 45
Ile Gln Leu Gln Ile Ser His Gln Leu Cys Asn Glu Ser Ser Arg Pro
50 55 60
Met Val Ser Val Ile Val Ala Lys Glu Glu Pro Met Asn Pro Ser Ser
65 70 75 80
Gln Val Val Cys Asp Asp Asp Pro Lys Ser Ile Phe Ser Ser Val Phe
85 90 95
Glu Glu Glu Pro Ile Val Leu Glu Lys His Ala Asn Gly Phe Leu Cys
100 105 110
Asp Ala Thr Pro Val Gln Ser Val Asp Cys Lys Leu Gln Asp Lys Asp
115 120 125
Glu Lys Ala Leu Val Leu Ala Gly Pro His Glu Leu Lys Ala Leu His
130 135 140
Leu Leu Lys Gly Asp Leu Lys Arg Glu Val Val Phe Cys Met Ser Phe
145 150 155 160
Val Gln Gly Asp Asp Ser Asp Asp Lys Ile Pro Val Thr Leu Gly Ile
165 170 175
Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Asp Thr Pro
180 185 190
Thr Leu Gln Leu Glu Asp Val Asp Pro Lys Ser Tyr Pro Lys Arg Asp
195 200 205
Met Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Arg Val
210 215 220
Glu Phe Glu Ser Ala Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln
225 230 235 240
Ala Glu Gln Lys Pro Val Phe Leu Gly Asn Ser Lys Gly Arg Gln Asp
245 250 255
Ile Thr Asp Phe Thr Met Glu Val Leu Ser Pro
260 265
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Ala Glu Val Pro Lys Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser
1 5 10 15
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Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met
20 25 30
Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile
35 40 45
Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala
50 55 60
Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro
65 70 75 80
Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe Pro Phe
85 90 95
Ile Phe Glu Glu Glu Pro Ile Phe Phe Asp Thr Trp Asp Asn Glu Ala
100 105 110
Tyr Val His Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp
115 120 125
Ser Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala
130 135 140
Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met
145 150 155 160
Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu
165 170 175
Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp
180 185 190
Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys
195 200 205
Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn
210 215 220
Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr
225 230 235 240
Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly
245 250 255
Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser
260 265
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser
1 5 10 15
Gly Asn Glu Asp Asp Leu Phe Phe Asp Val Asp Gly Pro Lys Gln Met
20 25 30
Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Gly Gly Gly Ile
35 40 45
CA 02269085 1999-10-18
39
Gln Leu Gln Ile Ser His Glu His Tyr Asn Glu Gly Phe Arg Gln Ala
50 55 60
Val Ser Val Val Val Ala Met Glu Lys Leu Arg Lys Met Leu Val Pro
65 70 75 80
Cys Pro Gln Ile Phe Gln Asp Asn Asp Leu Ser Thr Leu Ile Pro Phe
85 90 95
Ile Phe Glu Glu Glu Pro Val Phe Leu Asp Thr Arg Asn Asn Asp Ala
100 105 110
Cys Val His Asp Ala Pro Val Arg Ser Leu His Cys Thr Leu Arg Asp
115 120 125
Ala Gln Leu Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala
130 135 140
Leu His Leu Gln Gly Gin Asp Leu Glu Gln Gln Val Val Phe Ser Met
145 150 155 160
Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu
165 170 175
Gly Leu Lys Ala Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp
180 185 190
Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys
195 200 205
Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn
210 215 220
Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr
225 230 235 240
Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Arg Gly Gly
245 250 255
Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser
260 265
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ala Thr Val Pro Glu Leu Asn Cys Glu Met Pro Pro Phe Asp Ser
1 5 10 15
Asp Glu Asn Asp Leu Phe Phe Glu Val Asp Gly Pro Gln Lys Met Lys
20 25 30
Gly Cys Phe Gln Thr Phe Asp Leu Gly Cys Pro Asp Glu Ser Ile Gln
35 40 45
Leu Gln Ile Ser Gln Gln His Ile Asn Lys Ser Phe Arg Gln Ala Val
50 55 60
Ser Leu Ile Val Ala Val Glu Lys Leu Trp Gln Leu Pro Val Ser Phe
65 70 75 80
CA 02269085 1999-10-18
Pro Trp Thr Phe Gln Asp Glu Asp Met Ser Thr Phe Phe Ser Phe Ile
85 90 95
Phe Glu Glu Glu Pro Ile Leu Cys Asp Ser Trp Asp Asp Asp Asp Asn
100 105 110
Leu Leu Val Cys Asp Val Pro Ile Arg Gln Leu His Tyr Arg Leu Arg
115 120 125
Asp Glu Gln Gln Lys Ser Leu Val Leu Ser Asp Pro Tyr Glu Leu Lys
130 135 140
Ala Leu His Leu Asn Gly Gln Asn Ile Asn Gln Gln Val Ile Phe Ser
145 150 155 160
Met Ser Phe Val Gln Gly Glu Pro Ser Asn Asp Lys Ile Pro Val Ala
165 170 175
Leu Gly Leu Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp
180 185 190
Gly Thr Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro
195 200 205
Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Val Lys
210 215 220
Ser Lys Val Glu Phe Glu Ser Ala Glu Phe Pro Asn Trp Tyr Ile Ser
225 230 235 240
Thr Ser Gln Ala Glu His Lys Pro Val Phe Leu Gly Asn Asn Ser Gly
245 250 255
Gln Asp Ile Ile Asp Phe Thr Met Glu Ser Val Ser Ser
260 265
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Ala Thr Val Pro Glu Pro Ile Asn Glu Val Met Ala Tyr Tyr Ser
1 5 10 15
Asp Glu Asn Glu Leu Leu Phe Glu Val Asp Gly Pro Lys Gln Met Lys
20 25 30
Ser Cys Thr Gln His Leu Asp Leu Gly Ser Met Gly Asp Gly Asn Ile
35 40 45
Gln Leu Gin Ile Ser His Gln Leu Tyr Asn Lys Ser Phe Arg Gln Val
55 60
Val Ser Val Ile Val Ala Met Glu Lys Leu Arg Ser Arg Ala Tyr Glu
65 70 75 80
His Val Phe Arg Asp Asp Asp Leu Arg Ser Ile Leu Ser Phe Ile Phe
85 90 95
Glu Glu Glu Pro Val Ile Phe Glu Thr Ser Ser Asp Glu Leu Leu Cys
100 105 110
CA 02269085 1999-10-18
41
Asp Ala Ala Val Gln Ser Val Lys Cys Lys Leu Gln Asp Arg Glu Gln
115 120 125
Lys Ser Leu Val Leu Asp Ser Pro Cys Val Leu Lys Ala Leu His Leu
130 135 140
Pro Ser Gln Glu Met Ser Arg Glu Val Val Phe Cys Met Ser Phe Val
145 150 155 160
Gln Gly Glu Glu Arg Asp Asn Lys Ile Pro Val Ala Leu Gly Ile Arg
165 170 175
Asp Lys Asn Leu Tyr Leu Ser Cys Val Lys Lys Gly Asp Thr Pro Thr
180 185 190
Leu Gln Leu Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met
195 200 205
Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu
210 215 220
Phe Glu Ser Val Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile
225 230 235 240
Glu Glu Lys Pro Val Phe Leu Gly Arg Phe Arg Gly Gly Gln Asp Ile
245 250 255
Thr Asp Phe Arg Met Glu Thr Leu Ser Pro
260 265
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Ala Thr Val Pro Glu Leu Thr Ser Glu Met Met Ala Tyr His Ser
1 5 10 15
Gly Asn Glu Asn Asp Leu Phe Phe Glu Ala Asp Gly Pro Asn Tyr Met
20 25 30
Lys Ser Cys Phe Gln Asp Leu Asp Leu Cys Cys Pro Asp Glu Gly Ile
35 40 45
Gln Leu Arg Ile Ser Cys Gln Pro Tyr Asn Lys Ser Phe Arg Gln Val
50 55 60
Leu Ser Val Val Val Ala Leu Glu Lys Leu Arg Gln Lys Ala Val Pro
65 70 75 80
Cys Pro Gln Ala Phe Gln Asp Asp Gly Leu Arg Thr Phe Phe Ser Leu
85 90 95
Ile Phe Glu Glu Glu Pro Val Leu Cys Asn Thr Trp Asp Asp Tyr Ser
100 105 110
Leu Glu Cys Asp Ala Val Arg Ser Leu His Cys Arg Leu Gln Asp Ala
115 120 125
Gln Gln Lys Ser Leu Val Leu Ser Gly Thr Tyr Glu Leu Lys Ala Leu
130 135 140
CA 02269085 1999-10-18
42
His Leu Asn Ala Glu Asn Leu Asn Gln Gln Val Val Phe Ser Met Ser
145 150 155 160
Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly
165 170 175
Leu Arg Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Asp Lys
180 185 190
Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Asn Arg Tyr Pro Lys Lys
195 200 205
Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Lys Asp Lys
210 215 220
Leu Glu Phe Glu Ser Ala Gin Phe Pro Asn Trp Tyr Ile Ser Thr Ser
225 230 235 240
Gln Thr Glu Tyr Met Pro Val Phe Leu Gly Asn Asn Ser Gly Gly Gln
245 250 255
Asp Leu Ile Asp Phe Ser Met Glu Phe Val Ser Ser
260 265
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ala Thr Val Pro Glu Leu Asn Cys Glu Ile Ala Ala Phe Asp Ser
1 5 10 15
Glu Glu Asn Asp Leu Phe Phe Glu Ala Asp Arg Pro Gln Lys Ile Lys
20 25 30
Asp Cys Phe Gln Ala Leu Asp Leu Gly Cys Pro Asp Glu Ser Ile Gln
35 40 45
Leu Gln Ile Ser Gln Gln His Leu Asp Lys Ser Phe Arg Lys Ala Val
50 55 60
Ser Leu Ile Val Ala Val Glu Lys Leu Trp Gln Leu Pro Met Ser Cys
65 70 75 80
Pro Trp Ser Phe Gln Asp Glu Asp Pro Ser Thr Phe Phe Ser Phe Ile
85 90 95
Phe Glu Glu Glu Pro Val Leu Cys Asp Ser Trp Asp Asp Asp Asp Leu
100 105 110
Leu Val Cys Asp Val Pro Ile Arg Gln Leu His Cys Arg Leu Arg Asp
115 120 125
Glu Gln Gln Lys Cys Leu Val Leu Ser Asp Pro Cys Glu Leu Lys Ala
130 135 140
Leu His Leu Asn Gly Gln Asn Ile Ser Gln Gln Val Val Phe Ser Met
145 150 155 160
Ser Phe Val Gln Gly Glu Thr Ser Asn Asp Lys Ile Pro Val Ala Leu
165 170 175
CA 02269085 1999-10-18
4 ~
43
Gly Leu Lys Gly Leu Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Gly
180 185 190
Thr Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro Lys
195 200 205
Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Val Lys Thr
210 215 220
Lys Val Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr
225 230 235 240
Ser Gln Ala Glu His Arg Pro Val Phe Leu Gly Asn Ser Asn Gly Arg
245 250 255
Asp Ile Val Asp Phe Thr Met Glu Pro Val Ser Ser
260 265
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Ala Ile Val Pro Glu Pro Ala Lys Glu Val Met Ala Asn Tyr Gly
1 5 10 15
Asp Asn Asn Asn Asp Leu Leu Phe Glu Ala Asp Gly Pro Lys Glu Met
20 25 30
Lys Cys Cys Thr Gln Asn Leu Asp Leu Gly Ser Leu Arg Asn Gly Ser
35 40 45
Ile Gln Leu Gln Ile Ser His Gln Leu Trp Asn Lys Ser Ile Arg Gln
50 55 60
Met Val Ser Val Ile Val Ala Val Glu Lys Pro Met Lys Asn Pro Ser
65 70 75 80
Ser Gln Ala Phe Cys Asp Asp Asp Gln Lys Ser Ile Phe Ser Phe Ile
85 90 95
Phe Glu Glu Glu Pro Ile Ile Leu Glu Thr Cys Asn Asp Asp Phe Val
100 105 110
Cys Asp Ala Asn Val Gln Ser Met Glu Cys Lys Leu Gln Asp Lys Asp
115 120 125
His Lys Ser Leu Val Leu Ala Gly Pro His Met Leu Lys Ala Leu His
130 135 140
Leu Leu Thr Gly Asp Leu Lys Arg Glu Val Val Phe Cys Met Ser Phe
145 150 155 160
Val Gln Gly Asp Asp Ser Asn Asn Lys Ile Pro Val Thr Leu Gly Ile
165 170 175
Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Asn Thr Pro
180 185 190
Thr Leu Gln Leu Glu Asp Ile Asp Pro Lys Arg Tyr Pro Lys Arg Asp
195 200 205
CA 02269085 1999-10-18
44
Met Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Arg Val
210 215 220
Glu Phe Glu Ser Ala Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln
225 230 235 240
Ala Glu Gln Lys Pro Val Phe Leu Gly Asn Ser Lys Gly Arg Gln Asp
245 250 255
Ile Thr Asp Phe Thr Met Glu Val Leu Ser Pro
260 265
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
ATGGCCAAAG TTCCAGACAT GTTTGAAGAC CTGAAGAACT GTTACAGTGA AAATGAAGAA 60
GACAGTTCCT CCATTGATCA TCTGTCTCTG AATCAGAAAT CCTTCTATCA TGTAAGCTAT 120
GGCCCACTCC ATGAAGGCTG CATGGATCAA TCTGTGTCTC TGAGTATCTC TGAAACCTCT 180
AAAACATCCA AGCTTACCTT CAAGGAGAGC ATGGTGGTAG TAGCAACCAA CGGGAAGGTT 240
CTGAAGAAGA GACGGTTGAG TTTAAGCCAA TCCATCACTG ATGATGACCT GGAGGCCATC 300
GCCAATGACT CAGAGGAAGA AATCATCAAG CCTAGGTCAG CACCTTTTAG CTTCCTGAGC 360
AATGTGAAAT ACAACTTTAT GAGGATCATC AAATACGAAT TCATCCTGAA TGACGCCCTC 420
AATCAAAGTA TAATTCGAGC CAATGATCAG TACCTCACGG CTGCTGCATT ACATAATCTG 480
GATGAAGCAG TGAAATTTGA CATGGGTGCT TATAAGTCAT CAAAGGATGA TGCTAAAATT 540
ACCGTGATTC TAAGAATCTC AAAAACTCAA TTGTATGTGA CTGCCCAAGA TGAAGACCAA 600
CCAGTGCTGC TGAAGGAGAT GCCTGAGATA CCCAAAACCA TCACAGGTAG TGAGACCAAC 660
CTCCTCTTCT TCTGGGAAAC TCACGGCACT AAGAACTATT TCACATCAGT TGCCCATCCA 720
AACTTGTTTA TTGCCACAAA GCAAGACTAC TGGGTGTGCT TGGCAGGGGG GCCACCCTCT 780
ATCACTGACT TTCAGATACT GGAAAACCAG GCGTAGGTCT GGAGTCTCAC TTGTCTCACT 840
TGTGCAGTGT TGACAGTT 858
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 807 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ATGGCAGAAG TACCTGAGCT CGCCAGTGAA ATGATGGCTT ATTACAGTGG CAATGAGGAT 60
CA 02269085 1999-10-18
GACTTGTTCT TTGAAGCTGA TGGCCCTAAA CAGATGAAGT GCTCCTTCCA GGACCTGGAC 120
CTCTGCCCTC TGGATGGCGG CATCCAGCTA CGAATCTCCG ACCACCACTA CAGCAAGGGC 180
TTCAGGCAGG CCGCGTCAGT TGTTGTGGCC ATGGACAAGC TGAGGAAGAT GCTGGTTCCC 240
TGCCCACAGA CCTTCCAGGA GAATGACCTG AGCACCTTCT TTCCCTTCAT CTTTGAAGAA 300
GAACCTATCT TCTTTGACAC ATGGGATAAC GAGGCTTATG TGCACGATGC ACCTGTACGA 360
TCACTGAACT GCACGCTCCG GGACTCACAG CAAAAAAGCT TGGTGATGTC TGGTCCATAT 420
GAACTGAAAG CTCTCCACCT CCAGGGACAG GATATGGAGC AACAAGTGGT GTTCTCCATG 480
TCCTTTGTAC AAGGAGAAGA AAGTAATGAC AAAATACCTG TGGCCTTGGG CCTCAAGGAA 540
AAGAATCTGT ACCTGTCCTG CGTGTTGAAA GATGATAAGC CCACTCTACA GCTGGAGAGT 600
GTAGATCCCA AAAATTACCC AAAGAAGAAG ATGGAAAAGC GATTTGTCTT CAACAAGATA 660
GAAATCAATA ACAAGCTGGA ATTTGAGTCT GCCCAGTTCC CCAACTGGTA CATCAGCACC 720
TCTCAAGCAG AAAACATGCC CGTCTTCCTG GGAGGGACCA AAGGCGGCCA GGATATAACT 780
GACTTCACCA TGCAATTTGT GTCTTCC 807
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TAGTGAATTC GAAGAATACA GTTC 24
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
ACGGGAATTC TCTGAAGAAG AGACGG 26
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GATTGAATTC CAACCGTCTC TTCTTC 26
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(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CATGGAATTC CCAGAAGAAG AGGAG 25
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CAGCCATGGC AACCGTACCT G 21
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
ATCAGTACCC TGGAGTCTGC 20
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GGATCAGCTG AAGTCCATC 19
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CA 02269085 1999-10-18
47
CGAATTCATG GATTTTGAGT CA 22
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
TTGAGAAGTG CTGATGTACC 20
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CAGCTTGGAG CTCCATGCTG 20
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
CTTAGTTGTG GCGCTGGATG 20
