CA2269085C - Rainbow trout il1.beta.; encoding nucleic acid; materials and methods - Google Patents

Abstract

Rainbow Trout Interleukin (IL)-1.beta. has been cloned and encoding nucleic acid is provided, along with variants and homologues. Nucleic acid may be used in production of the polypeptide. The polypeptide or encoding nucleic acid therefor may be used in fish to confer one or more cytokine activities.

Description

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 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., 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 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 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 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

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 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%, 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 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.

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.

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 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 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 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, 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 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 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 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 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.

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 WO 98/17802' PCT/GB97/02855 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 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 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 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 low level expression of IL10 was detectable in kidney macrophages, expression was increased following stimulation with LPS.

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.

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.

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%

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:

Met Asp Phe Glu Ser Asn Tyr Ser Leu Ile Lys Asn Thr Ser Glu Ser Ala Ala Trp Ser Ser Lys Leu Pro Gin Gly Leu Asp Leu Glu Val Ser His His Pro Ile Thr Met Arg His Ile Ala Asn Leu Ile Ile Ala Met Glu Arg Leu Lys Gly Gly Glu Gly Val Thr Met Gly Thr Glu Phe Lys Asp Lys Asp Leu Leu Asn Phe Leu Leu Glu Ser Ala Val Glu Glu His Ile Val Leu Glu Leu Glu Ser Ala Pro Pro Ala Ser Arg Arg Ala Ala Gly Phe Ser Ser Thr Ser Gin Tyr Glu Cys Ser Val Thr Asp Ser Glu Asn Lys Cys Trp Val Leu Met Asn Glu Ala Met Glu Leu His Ala Met Met Leu Gin Gly Gly Ser Ser Tyr His Lys Val His Leu Asn Leu Ser Ser Tyr Val Thr Pro Val Pro Ile Glu Thr Glu Ala Arg Pro Val Ala Leu Gly Ile Lys Gly Ser Asn Leu Tyr Leu Ser Cys Ser Lys Ser Gly Gly Arg Pro Thr Leu His Leu Glu Glu Val Ala Asp Lys Asp Gin Leu Lys Ser Ile Ser Gin Gin Ser Asp Met Val Arg Phe Leu Phe Tyr Arg Arg Asn Thr Gly Val Asp Ile Ser Thr Leu Glu Ser Ala Ser Phe Arg Asn Trp Phe Ile Ser Thr Asp Met Gin Gin Asp Tyr Thr Lys Pro Val Asp Met Cys Gin Lys Ala Ala Pro Asn Arg Leu Thr Thr Phe Thr Ile Gln Arg His Asn (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 Ala Ala Trp Ser Ser Lys Leu Pro Gln Gly Leu Asp Leu Glu Val Ser His His Pro Ile Thr Met Arg His Ile Ala Asn Leu Ile Ile Ala Met Glu Arg Leu Lys Gly Gly Glu Gly Val Thr Met Gly Thr Glu Phe Lys Asp Lys Asp Leu Leu Asn Phe Leu Leu Glu Ser Ala Val Glu Glu His Ile Val Leu Glu Leu Glu Ser Ala Pro Pro Ala Ser Arg Arg Ala Ala Gly Phe Ser Ser Thr Ser Gln Tyr Glu Cys Ser Val Thr Asp Ser Glu Asn Lys Cys Trp Val Leu Met Asn Glu Ala Met Glu Leu His Ala Met Met Leu Gln Gly Gly Ser Ser Tyr His Lys Val His Leu Asn Leu Ser Ser Tyr Val Thr Pro Val Pro Ile Glu Thr Glu Ala Arg Pro Val Ala Leu Gly Ile Lys Gly Ser Asn Leu Tyr Leu Ser Cys Ser Lys Ser Gly Gly Arg Pro Thr Leu His Leu Glu Glu Val Ala Asp Lys Asp Gln Leu Lys Ser Ile Ser Gln Gln Ser Asp Met Val Arg Phe Leu Phe Tyr Arg Arg Asn Thr Gly Val Asp Ile Ser Thr Leu Glu Ser Ala Ser Phe Arg Asn Trp Phe Ile Ser Thr Asp Met Gln Gln Asp Tyr Thr Lys Pro Val Asp Met Cys Gln Lys Ala Ala Pro Asn Arg Leu Thr Thr Phe Thr Ile Gln Arg His Asn (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 Asp Glu Asn Glu Leu Leu Phe Glu Ala Asp Asp Pro Lys Gln Met Lys Ser Cys Ile Gln His Leu Asp Leu Gly Ser Met Gly Asp Gly Asn Ile Gln Leu Gln Ile Ser His Gln Phe Tyr Asn Lys Ser Phe Arg Gln Val Val Ser Val Ile Val Ala Met Glu Lys Leu Arg Asn Ser Ala Tyr Ala His Val Phe His Asp Asp Asp Leu Arg Ser Ile Leu Ser Phe Ile Phe Glu Glu Glu Pro Val Ile Phe Glu Thr Ser Ser Asp Glu Phe Leu Cys Asp Ala Pro Val Gln Ser Ile Lys Cys Lys Leu Gln Asp Arg Glu Gln Lys Ser Leu Val Leu Ala Ser Pro Cys Val Leu Lys Ala Leu His Leu Leu Ser Gln Glu Met Asn Arg Glu Val Val Phe Cys Met Ser Phe Val Gln Gly Glu Glu Arg Asp Asn Lys Ile Pro Val Ala Leu Gly Ile Lys Asp Lys Asn Leu Tyr Leu Ser Cys Val Lys Lys Gly Asp Thr Pro Thr Leu Gln Leu Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu Phe Glu Ser Val Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile Glu Glu Arg Pro Val Phe Leu Gly His Phe Arg Ala Gly Gln Asp Ile Thr Asp Phe Arg Met Glu Thr Leu Ser Pro (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 Ser Asn Glu Asp Asp Leu Phe Phe Glu Val Asp Gly Pro Lys Gln Met Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile Gln Leu Gln Ile Ser His Glu His Tyr Asn Lys Gly Phe Arg Gln Ala Val Ser Val Val Val Ala Met Glu Lys Leu Arg Lys Met Leu Val Pro Cys Pro Gln Thr Phe Gln Asp Asn Asp Leu Ser Thr Leu Ile Pro Phe Ile Phe Glu Glu Glu Pro Val Phe Leu Asp Thr Cys Asn Asn Asp Ala Cys Val His Asp Ala Pro Val Arg Ser Leu His Cys Thr Leu Arg Asp Ala Gln Leu Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln Gly Gin Asp Val Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Val Pro Val Ala Leu Gly Leu Lys Ala Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Giu Ile Asn Asn Lys Leu Glu Cys Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Arg Gly Gly Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser (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 Asp Glu Asn Glu Leu Leu Phe Glu Ala Asp Gly Pro Lys Gln Met Lys Ser Cys Ile Gln His Leu Asp Leu Gly Ser Val Glu Val Gly Asn Ile Gln Leu Gln Ile Ser His Gln Leu Tyr Asn Lys Ser Phe Arg Gln Val Val Ser Val Ile Val Ala Met Glu Lys Leu Arg Asn Ser Ala Tyr Ala His Val Phe His Asp Asp Asp Leu Arg Asn Val Leu Ser Phe Ile Phe Glu Glu Glu Pro Val Ile Phe Glu Thr Ser Ser Asp Glu Phe Leu Cys Asp Ala Ala Val Gln Ser Val Asn Cys Lys Leu Gln Asp Arg Glu Gln Asn Ser Leu Val Leu Ala Ser Pro Cys Val Leu Lys Ala Leu His Leu Leu Ser Gln Glu Met Ser Arg Glu Val Val Phe Cys Met Ser Phe Val Gln Ala Glu Glu Arg Asp Asn Lys Ile Pro Val Ala Leu Gly Ile Arg Asp Lys Asn Gln Tyr Leu Ser Cys Val Lys Lys Gly Asp Thr Pro Thr Leu Gln Leu Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asp Thr Val Glu Phe Glu Ser Val Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser His Pro Glu Glu Lys Pro Val Phe Leu Gly His Phe Arg Gly Gly Gln Asp Ile Thr Asp Phe Arg Met Glu Thr Leu Ser Pro (2) INFORMATION FOR SEQ ID NO:6:

(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 Asp Glu Asn Asp Leu Phe Phe Glu Ala Asp Gly Pro Glu Lys Met Lys Gly Ser Leu Gln Asn Leu Ser His Ser Phe Leu Gly Asp Glu Gly Ile Gln Leu Gln Ile Ser His Gln Pro Asp Asn Lys Ser Leu Arg His Ala Val Ser Val Ile Val Ala Met Glu Lys Leu Lys Lys Ile Ser Phe Ala Cys Ser Gln Pro Leu Gln Asp Glu Asp Leu Lys Ser Leu Phe Cys Cys Ile Phe Glu Glu Glu Pro Ile Ile Cys Asp Thr Trp Asp Asp Gly Phe Val Cys Asp Ala Ala Ile Gln Ser Gln Asp Tyr Thr Phe Arg Asp Ile Ser Gln Lys Ser Leu Val Leu Ser Gly Ser Tyr Glu Leu Arg Ala Leu His Leu Asn Gly Gln Asn Met Asn Gln Gln Val Val Phe Arg Met Ser Phe Val His Gly Glu Glu Asn Ser Lys Lys Ile Pro Val Val Leu Cys Ile Lys Lys Asn Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Gly Lys Pro Thr Leu Gln Leu Glu Met Leu Asp Pro Lys Val Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Thr Glu Ile Lys Gly Asn Val Glu Phe Glu Ser Ser Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu Glu Met Pro Val Phe Leu Gly Asn Thr Lys Gly Gly Gln Asp Ile Thr Asp Phe Ile Met Glu Ser Ala Ser (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Ala Thr Val Pro Glu Pro Ala Lys Glu Val Met Ala Asn Asn Gly Asp Asn Asn Asn Asp Leu Leu Phe Glu Ala Asp Gly Pro Lys Glu Met Lys Cys Arg Thr Gln Asn Leu Asp Leu Ser Pro Leu Gly Asp Gly Ser Ile Gln Leu Gln Ile Ser His Gln Leu Cys Asn Glu Ser Ser Arg Pro Met Val Ser Val Ile Val Ala Lys Glu Glu Pro Met Asn Pro Ser Ser Gln Val Val Cys Asp Asp Asp Pro Lys Ser Ile Phe Ser Ser Val Phe Glu Glu Glu Pro Ile Val Leu Glu Lys His Ala Asn Gly Phe Leu Cys Asp Ala Thr Pro Val Gln Ser Val Asp Cys Lys Leu Gln Asp Lys Asp Glu Lys Ala Leu Val Leu Ala Gly Pro His Glu Leu Lys Ala Leu His Leu Leu Lys Gly Asp Leu Lys Arg Glu Val Val Phe Cys Met Ser Phe Val Gln Gly Asp Asp Ser Asp Asp Lys Ile Pro Val Thr Leu Gly Ile Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Asp Thr Pro Thr Leu Gln Leu Glu Asp Val Asp Pro Lys Ser Tyr Pro Lys Arg Asp Met Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Arg Val Glu Phe Glu Ser Ala Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu Gln Lys Pro Val Phe Leu Gly Asn Ser Lys Gly Arg Gln Asp Ile Thr Asp Phe Thr Met Glu Val Leu Ser Pro (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 Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe Pro Phe Ile Phe Glu Glu Glu Pro Ile Phe Phe Asp Thr Trp Asp Asn Glu Ala Tyr Val His Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser (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 Gly Asn Glu Asp Asp Leu Phe Phe Asp Val Asp Gly Pro Lys Gln Met Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Gly Gly Gly Ile Gln Leu Gln Ile Ser His Glu His Tyr Asn Glu Gly Phe Arg Gln Ala Val Ser Val Val Val Ala Met Glu Lys Leu Arg Lys Met Leu Val Pro Cys Pro Gln Ile Phe Gln Asp Asn Asp Leu Ser Thr Leu Ile Pro Phe Ile Phe Glu Glu Glu Pro Val Phe Leu Asp Thr Arg Asn Asn Asp Ala Cys Val His Asp Ala Pro Val Arg Ser Leu His Cys Thr Leu Arg Asp Ala Gln Leu Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln Gly Gin Asp Leu Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Ala Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Arg Gly Gly Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser (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 Asp Glu Asn Asp Leu Phe Phe Glu Val Asp Gly Pro Gln Lys Met Lys Gly Cys Phe Gln Thr Phe Asp Leu Gly Cys Pro Asp Glu Ser Ile Gln Leu Gln Ile Ser Gln Gln His Ile Asn Lys Ser Phe Arg Gln Ala Val Ser Leu Ile Val Ala Val Glu Lys Leu Trp Gln Leu Pro Val Ser Phe Pro Trp Thr Phe Gln Asp Glu Asp Met Ser Thr Phe Phe Ser Phe Ile Phe Glu Glu Glu Pro Ile Leu Cys Asp Ser Trp Asp Asp Asp Asp Asn Leu Leu Val Cys Asp Val Pro Ile Arg Gln Leu His Tyr Arg Leu Arg Asp Glu Gln Gln Lys Ser Leu Val Leu Ser Asp Pro Tyr Glu Leu Lys Ala Leu His Leu Asn Gly Gln Asn Ile Asn Gln Gln Val Ile Phe Ser Met Ser Phe Val Gln Gly Glu Pro Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Gly Thr Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Val Lys Ser Lys Val Glu Phe Glu Ser Ala Glu Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu His Lys Pro Val Phe Leu Gly Asn Asn Ser Gly Gln Asp Ile Ile Asp Phe Thr Met Glu Ser Val Ser Ser (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 Asp Glu Asn Glu Leu Leu Phe Glu Val Asp Gly Pro Lys Gln Met Lys Ser Cys Thr Gln His Leu Asp Leu Gly Ser Met Gly Asp Gly Asn Ile Gln Leu Gin Ile Ser His Gln Leu Tyr Asn Lys Ser Phe Arg Gln Val Val Ser Val Ile Val Ala Met Glu Lys Leu Arg Ser Arg Ala Tyr Glu His Val Phe Arg Asp Asp Asp Leu Arg Ser Ile Leu Ser Phe Ile Phe Glu Glu Glu Pro Val Ile Phe Glu Thr Ser Ser Asp Glu Leu Leu Cys Asp Ala Ala Val Gln Ser Val Lys Cys Lys Leu Gln Asp Arg Glu Gln Lys Ser Leu Val Leu Asp Ser Pro Cys Val Leu Lys Ala Leu His Leu Pro Ser Gln Glu Met Ser Arg Glu Val Val Phe Cys Met Ser Phe Val Gln Gly Glu Glu Arg Asp Asn Lys Ile Pro Val Ala Leu Gly Ile Arg Asp Lys Asn Leu Tyr Leu Ser Cys Val Lys Lys Gly Asp Thr Pro Thr Leu Gln Leu Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu Phe Glu Ser Val Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile Glu Glu Lys Pro Val Phe Leu Gly Arg Phe Arg Gly Gly Gln Asp Ile Thr Asp Phe Arg Met Glu Thr Leu Ser Pro (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 Gly Asn Glu Asn Asp Leu Phe Phe Glu Ala Asp Gly Pro Asn Tyr Met Lys Ser Cys Phe Gln Asp Leu Asp Leu Cys Cys Pro Asp Glu Gly Ile Gln Leu Arg Ile Ser Cys Gln Pro Tyr Asn Lys Ser Phe Arg Gln Val Leu Ser Val Val Val Ala Leu Glu Lys Leu Arg Gln Lys Ala Val Pro Cys Pro Gln Ala Phe Gln Asp Asp Gly Leu Arg Thr Phe Phe Ser Leu Ile Phe Glu Glu Glu Pro Val Leu Cys Asn Thr Trp Asp Asp Tyr Ser Leu Glu Cys Asp Ala Val Arg Ser Leu His Cys Arg Leu Gln Asp Ala Gln Gln Lys Ser Leu Val Leu Ser Gly Thr Tyr Glu Leu Lys Ala Leu His Leu Asn Ala Glu Asn Leu Asn Gln Gln Val Val Phe Ser Met Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Arg Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Asp Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Asn Arg Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Lys Asp Lys Leu Glu Phe Glu Ser Ala Gin Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Thr Glu Tyr Met Pro Val Phe Leu Gly Asn Asn Ser Gly Gly Gln Asp Leu Ile Asp Phe Ser Met Glu Phe Val Ser Ser (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 Glu Glu Asn Asp Leu Phe Phe Glu Ala Asp Arg Pro Gln Lys Ile Lys Asp Cys Phe Gln Ala Leu Asp Leu Gly Cys Pro Asp Glu Ser Ile Gln Leu Gln Ile Ser Gln Gln His Leu Asp Lys Ser Phe Arg Lys Ala Val Ser Leu Ile Val Ala Val Glu Lys Leu Trp Gln Leu Pro Met Ser Cys Pro Trp Ser Phe Gln Asp Glu Asp Pro Ser Thr Phe Phe Ser Phe Ile Phe Glu Glu Glu Pro Val Leu Cys Asp Ser Trp Asp Asp Asp Asp Leu Leu Val Cys Asp Val Pro Ile Arg Gln Leu His Cys Arg Leu Arg Asp Glu Gln Gln Lys Cys Leu Val Leu Ser Asp Pro Cys Glu Leu Lys Ala Leu His Leu Asn Gly Gln Asn Ile Ser Gln Gln Val Val Phe Ser Met Ser Phe Val Gln Gly Glu Thr Ser Asn Asp Lys Ile Pro Val Ala Leu 4 ~

Gly Leu Lys Gly Leu Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Gly Thr Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Val Lys Thr Lys Val Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu His Arg Pro Val Phe Leu Gly Asn Ser Asn Gly Arg Asp Ile Val Asp Phe Thr Met Glu Pro Val Ser Ser (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 Asp Asn Asn Asn Asp Leu Leu Phe Glu Ala Asp Gly Pro Lys Glu Met Lys Cys Cys Thr Gln Asn Leu Asp Leu Gly Ser Leu Arg Asn Gly Ser Ile Gln Leu Gln Ile Ser His Gln Leu Trp Asn Lys Ser Ile Arg Gln Met Val Ser Val Ile Val Ala Val Glu Lys Pro Met Lys Asn Pro Ser Ser Gln Ala Phe Cys Asp Asp Asp Gln Lys Ser Ile Phe Ser Phe Ile Phe Glu Glu Glu Pro Ile Ile Leu Glu Thr Cys Asn Asp Asp Phe Val Cys Asp Ala Asn Val Gln Ser Met Glu Cys Lys Leu Gln Asp Lys Asp His Lys Ser Leu Val Leu Ala Gly Pro His Met Leu Lys Ala Leu His Leu Leu Thr Gly Asp Leu Lys Arg Glu Val Val Phe Cys Met Ser Phe Val Gln Gly Asp Asp Ser Asn Asn Lys Ile Pro Val Thr Leu Gly Ile Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Asn Thr Pro Thr Leu Gln Leu Glu Asp Ile Asp Pro Lys Arg Tyr Pro Lys Arg Asp Met Glu Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Arg Val Glu Phe Glu Ser Ala Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu Gln Lys Pro Val Phe Leu Gly Asn Ser Lys Gly Arg Gln Asp Ile Thr Asp Phe Thr Met Glu Val Leu Ser Pro (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:

(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:

(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:

(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:

(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:

(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:

(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:

(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:

(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:

(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:

(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:

(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:

(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:

Claims (16)

1. An isolated polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

2. A polynucleotide according to claim 1 comprising the coding sequence shown in SEQ ID NO:1.

3. An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80%
identity with, and the same biological function as, the amino acid sequence shown in SEQ ID NO:2.

4. A polynucleotide according to claim 3 encoding a polypeptide comprising an amino acid sequence having at least 90% identity with, and the same biological function as, the amino acid sequence shown in SEQ ID NO:2.

5. A polynucleotide according to claim 3 encoding a polypeptide comprising an amino acid sequence having at least 95% identity with, and the same biological function as, the amino acid sequence shown in SEQ ID NO:2.

6. A polynucleotide according to any of claims 1 to 5 operably linked to a regulatory sequence for expression.

7. A nucleic acid vector suitable for transformation of a host cell and comprising a polynucleotide according to any one of claims 1 to 6.

8. A host cell containing a polynucleotide according to any one of claims 1 to 6 or nucleic acid vector according to claim 7.

9. A cell according to claim 8 which is a fish cell.

10. A method of producing a polypeptide encoded by a polynucleotide according to any one of claims 1 to 5, the method comprising expressing the polynucleotide according to claim 6 to produce the polypeptide.

11. A method according to claim 10 further comprising recovering the polypeptide.

12. A composition comprising a polypeptide encoded by the polynucleotide of any one of claims 1 to 6 and a pharmaceutically acceptable excipient, vehicle or carrier.

13. Use of a polynucleotide according to any of claims 1 to 6 for the production of the encoded polypeptide.

14. An isolated polypeptide encoded by a polynucleotide according to any of claims 1 to 6.

15. Use of a polypeptide according to claim 14, or a nucleic acid according to any one of claims 1 to 5 as an adjuvant for administration to a fish.

16. Use according to claim 15 wherein the nucleic acid includes a regulatory sequence for expression of the polypeptide.