T CELL STIMULATING PROTEIN OF PESTIVIRUS

Abstract: This application is concerned with immunogenic polypeptides of pestiviruses, specifically Classical Swine Fever Virus (CSFV), specifically the nonstructural protein p10, more specifically T cell epitopes from this protein and nucleic acid molecules encoding these polypeptides, and vaccines and diagnostics with these polypeptides or nucleic acid molecules.

Description: T CELL STIMULATING PROTEIN OF PESTIVIRUS This application is concerned with polypeptides comprising T lymphocyte stimulatory proteins of pestiviruses, especially Classical Swine Fever Virus (CSFV) , specifically the nonstructural protein plO, more specifically T cell epitopes from this protein, and nucleic acids coding for these polypeptides, recombinant vectors with these nucleic acids, host cells transformed with these vectors, vaccines and diagnostics with this polypeptides and processes for preparing them. The genus pestivirus belongs to the family of Flaviviridae and consists of Classical Swine Fever Virus (or hog cholera virus) , which is the causative agent of classical swinefever, bovine viral diarrhoea virus (BVDV) infectious to cattle and border disease virus (BDV) infectious to sheep. Pestiviruses are small enveloped RNA viruses having a diameter of 40-60 run. The virions consist of a nuclear capsid enveloped by a lipid layer embedded with glycoproteins. The genome of pestiviruses consists of a single stranded RNA of approximately 12.5 kB. It contains a single open reading frame (ORF) which is flanked by parts that are not being translated. The 3' end is not polyaαenyiated. Viral proteins are formed by co- and posttranslational processing of a hypothetic polyprotem, while the structural proteins are coded in the 5' end of the genome. In contrast to other flaviviridae the pestiviruses have a sequence coding for a non structural p23 protein at the Ξ' end. This N-termmal protease (Npro) is spliced from the next protein by an autoproteolytical process. In the 3' direction the sequence coding for a pl4 nuclear capsid and a signal sequence, which is responsible for the translocation of the subsequent sequences for glycoprotein EO, El and E2 in the lumen of the endoplasmatic reticulum (ER) , are following. The splicing of the single glycoproteins probably is caused by cellular SUBSTΓΠJTE SHEET (RULE 26) signalases n the ER. The glycoproteins can form complex structures in infected cells by dimensioning through S-S- bridges. The function of these complex structures is not yet known. Next to the sequences for the structural proteins the sequences for the non structural proteins pl25, plO, p30 and pl33 are* found in the polyprotein. Analogous to posttranslational processes in cytopathogenic Bovine Viral Diarrhea Virus (BVDV, another pestivirus) an 80p protein can be detected after processing the pl25 protein (Desport, M. and Brownlie J., Arch. Virol. Suppl . 3, 261-265, 1991) . The pl33 non structural protein, which is processed into p58 and p75 proteins, contains sequence motifs which resemble RNA polymerase sequences. The amino acid sequences of the non- structural CSFV proteins are considered to be approximately: 3- 1142 (pl25) , 1143-1206 (plO) shown in SEQ ID NO: 1 and 2 herein. The putative N-terminus of p80 is amino acid no. 460 (SEQ ID NO: 1 and 2) . The start of p30 is also shown in SEQ ID NO. 1 and 2, i.e. amino acid number 1207. The complete sequence of p30 for CSFV Alfort shown m Meyers et al . (Virology 171, 555-567, 1989; Fig. 4 ammo acids 2337-2683) . The DNA sequences encoding these proteins are also shown in SEQ ID NO. 1 herein and m Meyers et al . (supra) . On infection with CSFV acute, peracute, chronic or clinic invisible symptoms can occur. The severance of the disease is dependent on the infectious load on one hand, on the other hand the age of the animal, the immune competence and total constitution of the animal form important factors. In the peracute illness, which results in the dead of the animal after three to five days after infection with a highly virulent strain (e.g. the CSFV-Brescia strain) , only a fever is observable. In the acute disease state next to a high temperature, leucopenia, conjunctivitis and loss of appetite are observable. I the final stage cf the disease central nervous system disturbances occur. Further characteristics are an atrophy cf the thymus, cyanosis of the skin and cutaneous haemorrhages, partially caused by a thrombocytopenia. Mortality of the disease in the acute phase is 30-100%. The chronic disease form is found after infection with mesogenic virus strains. This form is most dangerous when piglets are infected in utero by diaplacental infection. After birth these piglets only survive for 6-8 weeks while they form a source of infection. Pigs, which survive a postnatal infection, gain lifetime immunity, which probably results from an induction of the humoral immune response. Neutralising antibodies are found after two to three weeks after infection. Several vaccines have been developed for this economically important disease. Vaccines with inactivated virus give only shortlasting protection and are not used anymore (Biront, P. and Leunen, J., in: Liess, B. (ed. ) : Classical swine fever and related viral infections. Martinus Nijhoff Publ. , Boston pp. 181-200, 1988) . Better protection has been established with attenuated viruses obtained by serial passaging in rabbits (C- strains) or in cell cultures (e.g. Thiverval-stram) (Launais, M. et al., Rev. Med. Vet., 123, 1537-1554, 1972; Shi i∑u Y., Jap. J. Trop. Agr . Res. Sci . , 13, 167-170, 1980) . However, a disadvantage of these vaccines is that tney do not discern between vaccinated animals ana animals infected with a field virus. In tne European Zommunity, tnerefcre, jse of tnese vaccines is forbidden presently ana contrc. c: classical swme fever is established by isolaticr. a a slaughtering of infected swine. In recent years some results have been obtained wit.-, -ive vaccines based on recombinant viruses (van Zijl, M. et a... , J. Virol. 65, 2761-2765, 1991; Rumenapf. T. et al . , J. Virc . i , 589-597, 1991; Hulst, M.M. et al . , J. Virol. 67, 5435-:-;.;:, 1993) . These vaccines have all been based on the expressicr. c: structural glycoproteins in recombinant vector viruses. The invention now is αirected to a polypeptide comprising a pestivirus T lymphocyte stimulatory protein, or an lmmunogenically active part thereof. Such a polypeptide is essentially free from other pestiviral proteins with which it is ordinarily associated. Specifically the T lymphocyte stimulatory protein is a Classical Swine Fever Virus T lymphocyte stimulatory protein, or an lmmunogenically active part thereof. More specifically the polypeptide comprises the CSFV non- structural plO protein. Because the CSFV proteins are expressed by the virus as a polyprotein which is cleaved subsequent to translation, also polypeptides comprising the plO protein and its flanking protein (s) pl25 and/or p30 or parts thereof are contemplated herein. More specifically the polypeptide comprises a T cell epitope with the ammo acid sequence S-T-A-E-N-A-L-L-V-A-L-F-G- Y-V, most specifically the polypeptide comprises a T cell epitope with the ammo acid sequence E-N-A-L-L-V-A-L-F. In general, the term "protein" refers to a molecular chain of amino acids with biological activity. A protein is not cf a specific length and can, if required, be modified in vivo cr in vitro, by, for example, glycosylation, amidation, carboxylation or phospnorylation; thus, inter alia, peptiαes, oligopept αes ana polypeptiaes are included within tne definition. More particularly, this invention provides polypeptides comprising T-lymphocyte stimulatory proteins, or lmmunogenically active parts tnereof, which comprise the ammo acid sequence shown in SEQ ID NO. 2 and their biologically functional equivalents or variants. lmmunogenically active parts cf these polypeptides are those parts of the ammo acid sequence that are able to e_ cιt a T cell activation. More specifically this invention includes polypeptides, which comprise the ammo acid sequence shown in SEQ ID NO: 6 and still more specifically are polypeptides which comprise the am o acid sequence snown in SEQ ID NO: 4. The biologically functional equivalents or variants of the proteins specifically disclosed herein are proteins derived from the above noted ammo acid sequences, for example by deletions, insertions and/or substitutions of one or more amino acids, but retain one or more immunogenic determinants of CSFV, i.e. said variants nave one or more epitopes capable of eliciting an immune response in a host animal. It will be understood that, for the particular proteins embraced herein, natural variations can exist between individual virus strains. These variations may be demonstrated by (an) ammo acid difference (s) in the overall sequence or by deletions, substitutions, insertions, inversions cr additions of (an) ammo acιd(s) in said sequence. Ammo acid substitutions whicn do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al in "The Proteins" Academic Press New York (1979j . Am o acid replacements oetween related ammo acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayncf, M.L., Atlas of protein sequence ana structure, Nat. Biomed. Res. Found., Washington D.C., 1976, vol. 5, suppl . 3) . Other ammo acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pre, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu. Baseα on this information, Lipman ana Pearson developed a methoα for rapiα and sensitive protein comparison (Science, 22~, 143Ξ-1441, 1985 1 and determining tne functional similarity oetween nomoloαous proteins. Sucr. amino ac d substitutions of tne exemplary emooαiments cf this invention are within tne scope cf tne invention as long as tne resulting proteins retain their immunoreactivity. The preparation of the proteins according to the invention is effected by means of one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques. The organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of a condensation reaction, either in homogenous phase or with the aid of a so-called solid phase. The most common methods for the above condensation reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method using activated esters, such as described in The Peptides, Analysis, Synthesis, Biology Vol. 1-3 (Ed.: Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc.) . Preparation of suitable fragments of above-mentioned peptides according to the invention using the "solid phase" is for instance described J. Amer . Che . Soc . 8_5, 2149 (1963) and Int. J. Peptide Protein Res. 3_5, 161-214 (1990) . The coupling cf the aminc acids of the peptide to be prepared usually starts from the carDoxyl end side. Fcr this method a solid pnase s needed on which there are reactive groups or or. which such groups can be introduced. This can be, fcr example, a copoly er of benzene and divmylbenzene with reactive chloromethyl groups, or a polymeric solid phase rendered reactive with hydroxy ethyl or amine-function. A particularly suitable solid phase is, for example, the p- alkoxybezyl alcohol resin (4-hydroxy-methyi-phenoxy-methyi- copolystyrene-1 - divmylbenzene resin, , described by Wang, J. Am. Chem. Soc. _9_5, 1326 (1974) . After synthesis the peptides can be split from th s solid phase under mild conditions. In order to determine if any protein and which was responsible for a T cell mediated effect, CSFV infected target cells were cultured m the following way: from a miniature swine ("NIH-Mimpig" ; MHC d / d haplotype) a kidney was removed and cut to pieces under sterile conditions. The organ pieces were rinsed with PBS and pieces were pipetted in a culture flask. They were incubated in culture medium to which a collagenase-dispase solution was added. The cells were rinsed from the tissue with PBS, pelleted and washed and further cultivated. For obtaining a stable, transformed cell line the cells were transformed with a plasmid having the sequence for the "large T" antigen of SV-40 (Southern, P. and Berg, P., J. Molec. Appl. Genetics., 1 , 327-341, 1982; Fanning, E., -J^ Virol. , 66, 1289-1293, 1992) . The MAX-cells thus obtained were selected by culturing with a neomycm analogon G418 (Boehringer) and tested for mycoplasma contamination (Mycoplasma Detection Kit, Boehringer Mannheim) . The MAX cells were cultured in DMEM and were infected with CSFV. To screen the genome of CSFV for epitopes recognized by virus specific T lymphocytes, autologous MAX cells were infected with Vaccinia virus/CSFV recombinants expressing different viral proteins. Successful infection of target cells with the recoitiDinants was routine!'-, checked by tne cytopatnogemc effect of Vaccinia virus. In addition tne expression of CSFV proteins was demonstrated by lmmunocytocnemical studies and western-blot analysis of infected MAX cells. Effector cells obtained from immunized miniature swine were cocultivated with Vaccinia virus recombinant mfecteα radiolabeleo targets (or Vaccinia virus wildtype infected targets as a control) at different effector to target ratios for 4 hours. After centrifugation 100 ± cf eacn supernatant were collected and tne cnromium release determined as cpm in a gamma radiation counter. Percent specific lysis was calculated by the formula: (exp. cpm - spont . cpm ) 100 %specific lysis = (total cpm - spont. cpm) Target cell controls were included in the following way: in order to measure spontaneous radioisotope release (spont. cpm) , target cells were incubated without effectors. In addition, the total chromium incorporation in target cells was determined (total cpm) . In this way CSFV-specifIC CTL were identified. For a further characterization of the epitopes responsible for the effects a series of overlapping peptides was synthesized. These peptides were loaded onto the MAX target cells by incubation in wells of round bottom plates for 1 hour. Effector cells were then added to achieve an effector to target cell ratio of 100:1. The plates were centrifuged and incubated and the specific lysis was determined by measuring chromium release as described above. According to a second aspect of the invention, there is provided a nucleic acid sequence encoding ail or a substantial part, in particular the immunologically active part, z: a purified pestivirus T lymphocyte stimulatory protein, more specifically a CSFV T cell stimulatory protein. A nucleic acid sequence according to the present mver.:::r. may be isolated from a particular CSFV strain and multiplier cy recombinant DNA techniques including poiymerase chain react cr. (PCR) technology cr may be chemically synthesized m vitr: cy techniques known the art. The invention further provides isolated and purified ' nucleic acid sequences encoding the above mentioned proteins of CSFV. Such nucleic acid sequences are shown SEQ. ID. NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. It is well known the art that the degeneracy of the genetic code permits substitution of bases in the codon resulting in another codon but still coding for the same amino acid, e.g. the codon for the ammo acid glutamic acid is both GAT and GAA. Consequently, it is clear that, for the expression of a protein with the amino acid sequence shown in SEQ. ID. NO: 2, the nucleic acid sequence may have a codon composition different from the nucleic acid sequence shown in SEQ. ID. NO: 1. Such a nucleic acid sequence may be operatively linked to various replication sequences with which it is not associated, or linked in nature, resulting in a so-called recombinant vector which can be used for the transfection of a suitable host. Useful recombinant vectors are preferably derived from plasmids, bacteriophages, cosmids or viruses. Specific vectors or cloning vehicles which can be used to clone nucleic acid sequences according to the invention are known in the art and include inter alia plasmid vectors such as pBR322, the various pUC, pGEM and Bluescript plasmids; bacteriophages, e.g. lambdagt-Wes, Charon 28 and the M13 derived phages or viral vectors such as SV4C, adenovirus or polyoma virus isee also Rodriguez, R. . and D.7. Denhardt, ed., Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988; Lenstra, J.A. et al., Arch. Virol . , 110, 1- 24, 1990) . The methods to be used fcr the construction of a recombinant vector according tc the invention are known to those of ordinary skill in the art and are inter alia set forth in Maniatis, T. et al . (Molecular Cloning A Laboratory Manual, second edition; Cold Spring Harbor Laboratory, 1989) . For example, the insertion cf the nuclei-: acid sequence according to the invention into a cloning vector can easily be achieved when both the qenes and the αesired clonmα vehicle have Deen cut with the same restriction enzyme (s) as complementary DNA termini are thereby produced. Alternatively, it may be necessary to modify the restriction sites that are produced into blunt ends either by digesting the single-stranded DNA or by filling m the single- stranded termini with an appropriate DNA polymerase. Subsequently, blunt end ligation with an enzyme such as T4 DNA ligase may be carried out. If desired, any restriction site may be produced by ligatmg linkers onto the DNA termini. Such linkers may comprise specific oligonucleotide sequences that encode restriction site sequences. The restriction enzyme cleaved vector and nucleic acid sequence may also be modified by homopolymeric tailing. "Transformation", as used herein, refers to the introduction of an heterologous nucleic acid sequence into a host cell, irrespective of the method used, for example direct uptake or transductio . The neterologous nucleic acid sequence may be maintained tnrougn autonomous replication or, alternatively, may oe integrated into the host genome. If desirec, tne recomomar.t vectors are prcviαeα witr. appropriate contrcl sequences compatible with the designated nost. These sequences can regulate the expression cf the inserted nucleic acid sequence. In addition to microorganisms, cell cultures derived from ulticellular organisms may also be used as hosts. The recombinant vectors according to the invention preferably contain one or more marker activities that may be used to select for desired transformantε, such as ampicill and tetracycline resistance pBR322, ampicill resistance and A-peptioe of β-gaiactosioase in pUC8. A suitable host cell is a microorganism or cell wnich can be transformed by a nucleic acio sequence encoding a polypeptide or by a recombinant vector comprising such a nucleic acid sequence, and which can, f desired, be used to express said polypeptide encoded by said nucleic acid sequence. The host cell can be of prokaryotic origin, e.g. bacteria such as Escherichia col , Bacillus subtilis and Pseudomonas species; or of eukaryotic origin such as yeasts, e.g. Saccharomyces cerevisiae or higher eukaryotic cells such as insect, plant or mammalian cells, including HeLa cells and Chinese hamster ovary (CHO) cells. Insect cells include the Sf9 cell line of Spodoptera frugiperda (Luckow et al . , Biotechnology 6 , 47-55, 1988) . Information with respect to the cloning and expression of the nucleic acid sequence of the present invention in eukaryotic cloning systems can be found in Esser, K. et al . (Plasmids of Eukaryotes, Sprmger-Verlag, 1986) . As host organism also other viruses can be used, which are able to express the inserted pestivirus sequence. Such viruses are commonly denoted vector viruses. In general, prokaryotεs are preferred for the construction of the recombinant vectors useful m tne present invention. E. coli K12 strains are particularly useful, especially DH5a or MC1061 strains. For expression, nucleic acid seαuences of the present invention are introduced into an expression vector, .e. said sequences are operably linked to expression control sequences. Such control sequences may comprise promotors, enhancers, operators, αucers, rioosome binding sites etc. Therefore, the present invention provides a recomomant vector comprising a nucleic acid sequence encoding a CSFV protein identified aoove operably linked tc expression control sequences, whicn is capable of expressing the DNA sequences contained therein (a) transformed host ceil(s) or organism. It should be understood, of course, that the nucleotide sequences inserted at the selected site of the cloning vector may include nucleotides which are not part of the actual structural gene for the desired polypeptide, or may include only a fragment of the complete structural gene for the desired protein as long as the transformed host will produce a polypeptide having at least one or more immunogenic determinants of a CSFV protein antigen. When the host cells are bacteria, useful expression control sequences which may be used include the Trp promotor and operator (Goeddel, et al., Nucl . Acids Res . , B_, 4057, 1980) ; the lac promotor and operator (Chang, et al . , Nature, 275, 615, 1978) ; the outer membrane protein promotor (Nakamura, K. and Inouge, M., EMBO J. , 1 , 771-775, 1982) ; the bacteriophage lambda promotors and operators (Remaut, E. et al. , Nucl. Acids Res. , 11, 4677-4688, 1983) ; the A-amylase (B. subtilis) promotor and operator, termination sequences and other expression enhancement and control sequences compatible with the selected host cell. When the host cell is yeast, illustrative useful expression control sequences include, e.g., A-mating factor. For insect cells the polyhedrm or plO promotors of baculoviruses can be used (Smith, G.E. et al., Mol . Cell . Biol . 2 , 2156-65, 1983J . When tne nost ceil is of mammalian origin illustrative useful expression control sequences include the SV-40 promotor (Ber an, P.W. et al . , Science, 222, 524-527, 1983) or the metallothionein promotor (Brmster, R.L., Nature, 296, 39-42, 1982) or a heat snock promotor (Voellmy et al . , Proc. Natl . Acad. Sci. USA, 82, 4949- 53, 1985) . Alternatively, expression control sequences present m CSFV may also be applied. For maximizing gene expression, see also Roberts and Lauer (Methods Enzymology, 65, " '3, 1979) . Therefore, the invention also comprises ;ai host cell(s or organιsm(s) having a nucleic acid sequence or a recombinant nucleic acid molecule or a recombinant vector described above, capable of producing the pestivirus protein by expression of the nucleic acid sequence. Immunization of animals against pestivirus infection, especially swine against CSFV, can be achieved by administering to the animals a polypeptide according to the invention in an lmmunologically relevant context as a so-called subunit vaccine. The subunit vaccine according to the invention may comprise a polypeptide in a pure form, optionally in the presence of a pharmaceutically acceptable carrier. The polypeptide can optionally be covalently bonded to a non- related protein, which can be of advantage in the purification of the fusion product. Examples are β-galactosidase, protein A, prochymosine, blood clotting factor Xa, etc. In some cases the ability to raise protective immunity using these polypeptides per se may be low. Small fragments are preferably conjugated to carrier molecules in order to raise their lmmunogenicity. Suitable carriers for this purpose are macromolecules, such as natural polymers (proteins like key hole limpet hemocyanin, albumin, toxins) , synthetic polymers like polyammo acids (polyiysme, polyalanme, , or micelles of amphiphiii compounds like saponins and paimitinic acid. Alternatively these fragments may be provided as polymers thereof, preferably linear polymers. If required, the proteins according to the invention which are to be used in a vaccine can be modified m vitro or m vivo, for example by glycosylation, amidation, carboxylation or phosphorylation. The lmmunologicai system will even be more effectively- triggered when the vaccine comprises the polypeptides as presented m an MHC molecule by an antigen presenting cell (APC) . Antigen presentation can be achieved by using monocytes, macrophages, mterdigitating cells, angerhans cells and especially oendritic cells, loaded with one of the peptides of the invention. Loading of the APC' s can be accomplished by bringing the polypeptides of the invention into or in the neighbourhood of the APC, but it is more preferable to let the APC process the complete antigen. In this way a presentation is achieved which mimicks the in vi vo situation the most realistic. Furthermore the MHC used by the cell is of the type which is suited to present the epitope. An overall advantage of using APC' s for the presentation of the epitopes is the choice of APC cell that is used in this respect. It is known from different types of APC' s that there are stimulating APC' s and inhibiting APC' s . Prefered are the listed cell types, which are so-called 'professional' antigen presenting cells, characterized in that they have co-stimulating molecules, which have an important function in the process of antigen presentation. Such co- stimulating molecules are, for example, B7, CTLA-4, CD70 or heat stable antigen. Fibroblasts, which have also been shown to be able to act as an antigen presenting cell, lack these co-stimulat g molecules . It is also possible to use cells already transfected with a cloning vehicle harbouring the information fcr the polypeptides of tne invention and which are cotransfecteo with a cloning vehicle which comprises the nucleotide sequence for an MHC molecule. These cells will act as an antigen presenting cell and will present pestivirus epitopes m the MHC molecules which are expressed on their surface. It is envisaged that this presentation will be enhanced, when the cell is also capable of expressing one of the above-mentioned co-stimulatmg molecules, or a molecule with a similar function. Th s expression can be the result cf transformation or transfection cf the cell with a third cloning vehicle having the sequence information coding for such a co-stimulat g molecule, but t can also be that the cell already was capable of production of co-stimulating molecules . In stead of a vaccine with these cells, which next to the desired expression products, also harbour many elements which are also expressed and which can negatively affect the desired immunogenic reaction of the cell, it is also possible that a vaccine is composed with liposomes which expose MHC molecules loaded with peptides, and which, for instance, are filled with lymphokines. Such liposomes will trigger a lmmunologic T cell reaction. By presenting the peptide the same way as it is also presented in vi vo an enhanced T cell response will be evoked. Furthermore, by the natural adjuvant working of the, relatively large, antigen presenting cells also a B cell response is triggered. This B cell response will a.o. lead to the formation of antibodies directed to the peptide-MHC complex. It is this naturally occurring phenomenon which is enlarged by the vaccination of APC' s already presenting the peptides of the invention. By enlarging not only an enlarged T cell response will be evoked, but also a B cell response which leads to antibodies directed to the MHC-peptide complex will be initiated. An alternative tc suounit vaccines is live vaccines. A nucleic acic sequence according tc tne invention is troαuceα by recombinant DNA tecnniques into a noεt cell or organisr (e.g. a bacterium or virus) in such a way that tne recombinant host is still able to replicate, thereby expressing a polypeptide coded by the inserted nucleic acid sequence and eliciting an immune response in the infected nost swine. A preferred emoodiment of the present invention is a recombinant vector virus comprising a neterclogous nucleic acic sequence descrioeα above, capable cf expressing the DNA sequence in (a) nost cell(s cr host swine infected witr. the recombinant vector virus. The term "heterologous" indicates that the nucleic acid sequence according to the invention is not normally present in nature in the vector virus. Furthermore, the invention also comprises (a) host cell(s) or cell culture infected with the recombinant vector virus, capable of producing the CSFV protein by expression of the nucleic acid sequence. For example the well known technique of in vivo homologous recombination can be used to introduce an heterologous nucleic acid sequence according to the invention into the genome of the vector virus. First, a DNA fragment corresponding with an insertion region of the vector genome, i.e. a region which can be used for the incorporation of an heterologous sequence without disrupting essential functions of the vector such as those necessary for infection or replication, is inserted into a cloning vector according to standard recDNA techniques. Insertion-regions have been reported for a large number of microorganisms (e.g. EP 80,806, EP 110,385, EP 83,286, EP 314,569, WO 88/02022, WC 88/07086, US *;,769,330 anc US 4,722, 848) . Second, f desired, a deletion can be introduced intc the insertion region present the recombinant vector molecule obtained from the first step. This can be achieved for example by appropriate exonuclease III digestion or restriction enzyme treatment of the recombinant vector molecule from the first step. Third, the heterologous nucleic acid sequence is inserted into the insertion-region present in the recombinant vectcr of the first step cr in place of the DNA deleted from said recombinant vector. The insertion region DNA sequence snouic oe of appropriate length as to allow homologous recombination with the vector αenome to occur. Thereafter, suitable cells car. be infected with wild-type vector virus or transformed witn vector genomic DNA in the presence of the recombinant vector containing the insertion flanked by appropriate vector DNA sequences whereby recoitiDmation occurs between the corresponding regions m the recombinant vector and the vector genome. Recombinant vector progeny can now be produced in cell culture and can be selected for example genotypically or phenotypically, e.g. by hybridization, detecting enzyme activity encoded by a gene co-mtegrateo along with the heterologous nucleic acid sequence, or detecting the antigenic heterologous polypeptide expressed by the recombinant vector immunologically. Next, these recomoinant microorganisms can be administered to swine for immunization whereafter they maintains themselves for some time, or even replicate in the body of the inoculated animal, expressing in vivo a polypeptide coded for by the inserted nucleic acid sequence according to the invention resulting in the stimulation of the immune system of the inoculated animal. Suitable vectors for the incorporation of a nucleic acid sequence according to the invention can be derived from viruses such as pox viruses, e.g. vaccinia virus (EP 110,385, EP 83,286, US 4,769,330 and US 4,722 848,, herpes viruses such as Auιesz y virus (van Zijl, K . et al., J. Virol. 6_2(6), 2191-2195, 1988/, porcine respiratory syncitiae virus, aoeno virus or influenza virus, or bacteria sucn as E. col , Streptococcus suis, Actinooacillus pleuropneumoniae cr specific Salmonella species. With recombinant microorganisms of this type, the polypeptide synthesized m the nost animal can be exposed as a surface antigen. In tnis context fusion of the polypeptide with OMP proteins, or pilus proteins cf fcr example E. coli cr synthetic provision of signa± and anchcr sequences which are recognized by tne organism are conceivable. It is also possible that the pestivirus po ypeptiαe, if αesireα as part of a larger whole, is released siαe the animal to be immunized. In all cf tnese cases it is also possible that one or more immunogenic products will find expression whicn generate protection against various pathogens and/or against various antigens of a given pathogen. A vector vaccine according to the invention can be prepared by culturmg a recombinant bacterium or a host cell infected with a recombinant vector comprising a nucleic acid sequence according to the invention, whereafter recombinant bacteria or vector containing cells and/or recombinant vector viruses grown m the cells can be collected, optionally m a pure form, and formed into a vaccine optionally in a lyophilised form. Host cells transformed with a recombinant vector according to the invention can also be cultured under conditions which are favourable for the expression of a polypeptide coded by said nucleic acid sequence. Vaccines may be prepared using samples of the crude culture, nost cell lysates or host cell extracts, although in another embodiment more purified polypeptides according tc the invention are formed into a vaccine, depending on its intended use. In order to purify the polypeptides roouceα, host cells transformed with a recombinant vectcr according to the invention are cultured m an adequate volume and the polypeptides produced are isolated from sucr. ceils, cr from tne medium if the protein s excreted. Polypeptides excreted into the medium can oe isolated and purified by standard techniques, e.g. salt fractionation, centrifugation, ultraflitration, chromatograpn , gel filtration or immuno affinity chromatography, whereas intracellular polypeptides can be isolated by first collecting said cells, disrupting the cells, for example by sonication or by other mechanically disruptive means such as Frencn press, followed by separation cf tne ooiypeptioes from the otner intracellular components and forming tne polypeptiαes into a vaccine. Cell disruption could a so ce acnieved c_. cnemical e.g. EDTA or detergents such as Triton X114 cr enzymatic means, sucn as lvsozvme diσestion. IS It is also possible to vaccinate animals with the "nude" DNA, i.e. the nucleic acids as defined above without any regulatory sequences. This DNA then will be incorporated in the genome of the vaccinated animal and thus express the polypeptide of the invention. The vaccines according to the invention can be enriched by numerous compounds which have an enhancing effect on the initiation and the maintenance of both the T cell and the B cell response after vaccination. In this way addition of cytokines to the vaccine will enhance the T cell response. Suitable cytokines are for instance mterleukmes, such as IL-2, IL-4, IL-7, or IL-12, GM- CSF, RANTES, tumor necrosis factor and interferons, such as IFN-gamma. Antibodies or antiseru directed against a polypeptide according to the invention have a potential use in passive immunotherapy, diagnostic immunoassays and generation of anti- idiotypic antibodies. The vaccine according tc the invention can be administered m a conventional active immunization scneme: single or repeated administration in a manner compatible with the αosage formulation, and m such amount as will oe prophylacticaliy effective, i.e. the amount cf immunizing antigen or recombinant microorganism capable of expressing said antigen that will induce immunity in swine against challenge by virulent CSFV. It can also be given in addition to a conventional (B lymphocyte directed) vaccination to enhance the immunity caused by such a vaccination. Immunity is defined as the induction of a significant level of protection in a population of animals after vaccination compared to an unvacc ated group. For live viral vectcr vaccines the dose rate per animal may range from 10 2 -10 e pfu. A typical s ounit vaccine according tc the invention comprises 1 μg - 1 mg of the protein according to the invention . Such vaccines can be administered mtradermally, subcutaneously, intramuscularly, mtraperitoneally, intravenously, orally or mtranasally . Additionally the vaccine may also contain an aqueous medium or a water containing suspension, often mixed with other constituents m order to increase the activity and/or the snelf life. These constituents may be salts, pH buffers, stabilizers (such as skimmed milk or casein hydrolysate) , emulsifiers, adjuvants to improve the immune response (e.g. oils, muramyl dipeptide, aluminium. hydroxide, saponm, polyanions and amphipatic substances) and preservatives. It is clear that a vaccine according to the invention may also contain immunogens related to other pathogens of swine, or may contain nucleic acid sequences encoding these immunogens, like antigens of Actmobacillus pleuropneumoniae, Pseudorabies virus, Porcine Influenza virus, Porcine Parvovirus, Streptococcus suis, Transmissible Gastroenteritisvirus, Rotavirus, Escherichia coll, Erysipelothriy rhusiopathiae, Pasteurella multocida and Bordetella bronch septica. The invention also relates to an "lmmunocnemical reagent", which reagent comprises a protein according to the invention. The term "lmmunochemical reagent" signifies that tne protein according to the invention is bound to a suitaoie support cr is provided with a labelling substance. The supports that may be used are, for example, the inner wall of a microtest well or a cuvette, a tube or a membrane, filter, test strip or the surface cf a particle sucn as, for example, a latex particle, an erytnrccyte, a aye sc-, a metal sol or metal compound as sol particle. Labelling substances which can oe used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle. A nucleic acid sequence according to the invention can also be used to design specific probes for hybridization experiments for the detection of pestivirus related nucleic acids in any kind of tissue. The present invention also comprises a test kit comprising said nucleic acid sequence useful for the diagnosis of pestivirus, specifically CSFV infection. The invention also relates to a test kit to be used an immunoassay, this test kit containing at least one immunochemical reagent according to the invention. The immunochemical reaction which takes place using this test kit is preferably a sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction. For carrying out a sandwich reaction, the test k t can consist, for example, of a polypept αe according tc tne invention bonded to a solid support, for example the inner wall of a microtest well, and either a labelled poiypect oe according to the invention or a labelled anti-antioody. The invention is illustrated by the following examples. EXAMPLES EXAMPLE 1: Identification of CSFV specific cytotoxic lymphocytes. Peripheral blood leukocytes were isolated from inbred miniature swme of MHC d,/d haplotype after immunization with CSFV-strain Rie s (4-10 5 TCID50 l.m.) following repeated challenges with 2-10 7 TCID5-3 CSFV-stram Alfort and a final challenge with 3 ml intranasally of a serum (1*10*^ TCID50) obtained from a CSFV-strain Brescia infected pig. The cells were seeded in 96-well round bottom microtiter plates at a concentration of 1-2*10*^ cells per microculture in RPMI medium (10% FCS) and simultaneously restimulated with infectious virus (5-10 5 TCID 50 /ml CSFV-Alfort) for 3 to 5 days. Culturing of infected target cells was done in the following way: from a miniature swine ("NIH-Minipig" ; MHC ' d haplotype) a kidney was removed and cut to pieces under sterile conditions. The organ pieces were rinsed with PBS and 30-100 pieces were pipetted in a culture flask (2Ξ cm*- ) . They were incubated m culture medium (10 FCS) to which a collagenase- dispase solution (stocksolution of 2.5 mg/ml diluted 1:6 m medium' was added. The cells were rinsed from the tissue with PBS, pelleted (6 min, 750 g> and washed twice in PBS. They were cultivated DMEM (10 ' - FCS) in culture flasks (25 and 75 cm 2 ) . For obtaining a stable, transformed cell line cf target cells the kidney cells were transformed with a plasmid having the sequence for the "large T" antigen of SV-40 (Southern, P. and Berg, P., J. Molec. Appi . Genetics., 1_, 327-341, 1982; Fanning, E. , . Virol., 66, 1289-1292, 1992: . For this ourified SV-40 plasmid pSV-3 nee (from. Dr T.C. Mettenleiter, BFAV Tubingen) was mixed with iipofectm to a DNA-iipofectin ratic of 1:4 and incubated for 15 mm at room temperature. Of a largelv (80 c . ! confluent kidney cell culture the culture medium was removeα and 2 mi OptiMEM-Medium (Gibco' was added. The lipofectm/DNA suspension was then added dropwise and the culture was initially incubated for 12 hours at 37°c. Then the medium was replaced by cloning medium (DMEM, F10 (Gibco) and F12(Gibco) in a 2:1:1 ratio) with 10% FCS (foetal calf serum) and cultivated at 37°C. The MAX-cells thus obtained were selected by culturing with a neomycin analogon G418 (Boehringer) and tested for mycoplasma contamination (Mycoplasma Detection Kit, Boehringer Mannheim) . 1-10 6 target cells were infected with CSFV-Alfort at a m.o.i. of 0.5. 48 hours after infection the cells were trypsinated and collected in a 200 μl CTL assay medium (RPMI 1640 (Gibco) , 31 FCS/ . The target cells were labelled with 100 μCi of Na2 S1 CrO- j for 90 mm., washed three times and resuspended in CTL assay medium at a final concentration of 1*10 4 cells/ml. Effector cells were diluted to achieve different effector: target ratios and were added to triplicate wells in 100 μl volumes. l*10 3 /well target cells were added to the effector cells. The plates were centrifuged at lOOg for 5 minutes and incubated for 4 hours at 37° . After centrifugation at 600g fcr 10 minutes, 100 μl cf eacr. supernatant were collected and the chromium release determined as cpm a gamma radiation counter. Percent specific lysis was calculated by the formula: (exp. cpm - spont. cpm ) * 100 -sspecific lysis = (total cpm - spont. cpm) Target cell controls were included in the following way: in order to measure spontaneous radioisotope release tspont. cpm) , target cells were incubated without effectors. In addition, the total chromium incorporation in target cells was determined (total cpm) . Results Lysis of CSFV-mfected target cells by cytotoxic T lymphocytes was higher than in the control group (figure 1) . A more than 30% higher specific lysis was reached with a relatively low effector to target cell ratio (12:1), indicating a high rate of CSFV specific cytotoxic T lymphocytes. EXAMPLE 2: Cytotoxic T lymphocytes mediated lysis of target cells expressing truncated proteins. 1-10 6 MAX cells (obtained as described Example 1) were infected with different Vaccinia virus/CSFV recombmants at a multiplicity of infection of 2.0 for 16 hours. Figure 2 shows the relative positions of the proteins of CSFV and the identification of the Vaccinia virus/CSFV recombmants obtained. Cells which showed a moderate cytopathic effect, were trypsmated and collected in 200 μl CTL assay medium. The target cells were labelled with 100 μCi of a2 51 CrO-* for 90 mm, washed tnree times ana resuspended m CTL assay medium at a concentration of 1*10^* cells/ml prior tc use m the assay. The assay was conducted m the same way as describee Example 1. Results The results of the chromium release assays indicated that CSFV-spec fic CTL recognize only target cells infected wit tne Vaccinia vιrus/pl25S recombinant. Surprisingly, neither target cells expressing viral structural proteins nor cells expressing the autoprotease were lysed by the specific cytotoxic T lympnocytes (figure 3) . From the peptide products produced by the Vaccinia virus/pl25S recombinant it seemes that the 80 kD subunit or the plO protein are responsible for the recognition (figure 4, upper middle graph) . Narrowing down this area it appears that Vaccinia virus/CSFV recombinant p80/VZ still shows specific lysis, while p80/VA and p80/VX are ineffective. This means (see figure 2; the nucleotide positions refer to the sequence of CSFV Alfort shown by Meyers et al . , Virology 171, 555-567, 1989) that the T cell specific epitope is situated near the cleavage site between pl25 and plO. The region identified as the region which the epitope is harboured (the region still present in the recombinant p80/VZ and not present in p80/VA and p80/VX) is situated from ammo acid position 2223 to 2285 (according to the sequence of CSFV Alfort (Meyers et al . supra), i.e. ammo acid positions 1093 to 1155 of SEQ ID NO:2) . Data obtained by N-termmal protein sequencing of plO of BVDV- strain cp7 reveal that the cleavage site between pl25 and plO of CSFV-Alfort is located between ammo acid positions 2272 and 2273 of CSFV-Alfort, i.e. between nucleotide positions 3426 and 3427 of SEQ ID NO: 1. EXAMPLE 3: Identification cf a T cell epitope recognized by CΞFV-sDecific CTL. To determine the epitope recognized by virus specific CTL, nonapeptides and pentadecapeptides were syntnesized overlapping by 8 and 12 amino acids, respectively, covering the ammo acid region from positions 1093 to 1155 of SEQ ID NO:2. 1-10 6 target cells were labelled with 100 μCi of Na2 51 CrO-} for 90 mm, washed three times and resuspended in CTL assay medium at a final concentration of 2*10'- cells/ml. 50 μl of target cell suspension was added to triplicate wells cf 96 well round bottom plates. Tne target cells were loaded witr. peptide by incubation with 100 μl peptide solution (5 mg/ml stock solution m DMSO; 1:4000 to 1:500C dilutee m RPMI medium, resulting in approximately 50 to 125 ng/10 3 target cells) for 1 hour. 1-10 5 effector cells were added to each well in 50 μl volumes to achieve an effector to target cell ratio of 100:1. Chromium release assay was done in a way similar to Example 1 and 2. Results Specific lysis of target cells loaded with different peptides is shown in figures 5 and 6. In figure 5 it is shown that the nonapeptide E-N-A-L-L-V-A-L-F causes the largest percentage of specific lysis, while in figure 6 the pentadecapapetide S-T-A-E-N-A-L-L-V-A-L-F-G-Y-V resulted in a significantly higher lysis of target cells compared to the target cells incubated with other pentadecapeptides. This pentadecapapetide contains the sequence of the nonapeptide identified already m figure 5. Nevertheless, CTL lysed target cells loaded with the nonapeptide more efficiently than target cells loaded with the pentadecamer, suggesting that nonapeptides have a higher affinity to porcine MHC-I molecules than pentadecapeptides. EXAMPLE 4: Crossreactivity of CSFV-specifIC CTL. Experiments regarding the crossreactivity of CSFV-speci it CTL were performed using target cells infected with different CSFV-strams at a m.o.i. of 0.5 for 48 hours (performed ir. accordance to Example 1) or infected with the Vaccinia virus recombmants at a m.o.i. of 2.0 for 16 hours (performec ir. accordance tc Example 2) . The Vaccinia virus recombman s contained the coding sequences for the non-structural proteins pl25, plO and the N-termmal part of p30 of CΞFV-Aifcrt Tubingen (Vac-pl25S) or the corresponding proteins of BVDV strain cp7 (Vac-pl20S) . The CTL activity cf CSFV-sensitizeo effector cells was determined m chromium release assays using the infected MAX cells as targets. For control non-infecteα and vaccinia virus wildtype (Vac- WR) infected targets were used. Results The results of both the experiments with CSFV-strains and with vaccinia virus recombmants are depicted in Table I . From these results it can be seen that CSFV specific CTL were capable of lysing target cells infected with different CSFV- strains. It is also shown that CSFV specific CTL were capable of lysing target cells expressing nonstructural proteins of a BVDV-stram, thereby suggesting that these nonstructural proteins harbour a T cell specific epitope which may be conserved for pestiviruses. Table I . Cross-reactivity of CSFV-specific CTL. LEGENDS TO THE FIGURES Fig, Results of chromium release assay indicating CSFV specific lysis of target cells by cytotoxic T cells. Open circles indicate non infected control target cells, full circles indicate CSFV infected target cells. The 1 specific lysis is indicated on the Y-axis, while the X-axis shows the ratio of effector to target cells. Fig. 2: Vaccinia virus/CSFV recombmants used for infection of autologous target cells. The relative positions of the N- termmai autoprotease (NP ro ) , the structural proteins core (C) , E0, El, E2 and the nonstructural proteins pl25, p30 and pl33 on the CSFV polyprotein as well as their processing products are indicated. In addition, the viral proteins and truncated pl25- protems expressed as Vaccina virus/CSFV recombmants are shown. The ammo acid positions indicated are m accordance to the sequence of CSFV-Alfort. Fig. 3: CTL mediated lysis cf target cells infected with different Vaccinia virus/CSFV recomoinants . MAX-ceils were infected witn Vaccinia virus wildtype (Vacc-WR, open circles) and the Vaccinia virus/CSF recombmants Vacc-NP ro C (open squares) , Vacc-E012 (closed squares) cr Vacc-pl25Ξ (closed circles) and used as target cells in chromium release assays against CSFV- specific CTL. Axes identical to figure 1. Fig. 4: CTL mediated lysis cf target cells expressing truncated nonstructural proteins. Presentation cf data analog to figure 3. Fiα - . 5 : Lysis of peptide target cells by CSFV-specific CTL. Radiolabelled MAX-cells were incubated with different nonapeptides. The bars indicate percent specific lysis by an effector to target ratio of 100 to 1. Controls: specific lysis of CSFV-mfected and non-mfected target cells. Fig, Lysis of pentadecapeptide loaded target cells. Legends as in figure 5. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: AKZO NOBEL N.V. (B) STREET: Velperweg 76 (C) CITY: Arnhe (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP) : 6824 B (G) TELEPHONE: 04120-66204 (H) TELEFAX: 04120-50592 (I) TELEX: a pha nl (li) TITLE OF INVENTION: T cell stimulating protein of pestivirus (iii) NUMBER OF SEQUENCES: 6 (ιv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPC- (2) INFORMATION FOR SEQ ID NO: 1: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3639 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (i ) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (lv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Classical Swine Fever Virus (B) STRAIN: Alfort (vii) IMMEDIATE SOURCE: (B) CLONE: Alfl25 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..3639 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAG GGT GGT AAG ATA GAT GGT GGC TGG CAG AGA CAA CCG GTG ACC AGT 48 Lys Gly Gly Lys lie Asp Gly Gly Trp Gin Arg Gin Pro Val Thr Ser 1 10 15 TTT GAC ATC CAA CTC GCA CTG GCA GTC GTA GTA GTC GTT GTG ATG TTG 96 Phe Asp lie Gin Leu Ala Leu Ala Val Val Val Val Val Val Met Leu 20 25 30 CTG GCA AAG AGA GAC CCG ACT ACT TTC CCT TTG GTA ATC ACA GTG GCA 144 Leu Ala Lys Arg Asp Pro Thr Thr Phe Pro Leu Val lie Thr Val Ala 35 40 S ACC CTG AGA ACG GCC AAG ATA ACC AAC GGT TTT AGC ACA GAT CTA GTC 192 Thr Leu Arg Thr Ala Lys lie Thr Asn Gly Phe Ser Thr Asp Leu Val 50 55 60 ATA GCC ACA GTG TCG GCA GOT TTG TTA ACT TGG ACC TAT ATC AGC GAC 240 lie Ala Thr Val Ser Ala Ala Leu Leu Thr Trp Thr Tyr lie Ser Asp 65 70 75 80 TAC TAC AAA TAC AAG ACT TGG CTA CAG TAC CTC GTC AGC ACG GTG ACT 288 Tyr Tyr Lys Tyr Lys Tr.r Trp Leu Gin Tyr Leu Val Ser Thr Val Thr δ5 90 95 GGA ATC TTC CTG ATA AGG GTG CTG AAG GGA ATA GGC GAA TTG GAT CTG 33c Gly He Phe Leu He Arg Val Leu Lys Gly He Gly Glu Leu Asp Leu 100 105 110 CAC GCC CCA ACC TTG CCG TCT CAC AGA CCC CTC TTT TAC ATC CTT GTA 384 His Ala Pro Thr Leu Pro Ser His Arg Pro Leu Phe Tyr He Leu Val 115 120 125 TAC CTT ATT TCC ACT GCC GTG GTA ACT AGA TGG AAT CTG GAC GTA GCC 432 Tyr Leu He Ser Thr Ala Val Val Thr Arg Trp Asn Leu Asp Val Ala 130 135 140 GGA TTG TTG CTG CAG TGC GTC CCA ACT CTT TTA ATG GTT TTT ACG ATG 480 Gly Leu Leu Leu Gin Cys Val Pro Thr Leu Leu Met Val Phe Thr Met 145 150 155 160 TGG GCA GAC ATT CTC ACC CTA ATT CTC ATA CTA CCT ACT TAT GAG TTA 52B Trp Ala Asp He Leu Thr Leu He Leu He Leu Pro Thr Tyr Glu Leu 165 170 175 ACA AAG TTA TAC TAC CTT AAG GAA GTG AAG ATT GGG GCA GAA AGA GGT 576 Thr Lys Leu Tyr Tyr Leu Lys Glu Val Lys He Gly Ala Glu Arg Gly 180 185 190 TGG CTG TGG AAA ACT AAC TAT A J AGG GTA AAC GAC ATC TAC GAG GTC 624 Trp Leu Trp Lys T.-.r Asn Tyr Lys Arα Val Asn Asp lie Tyr G ^ _. Va. 195 20C 205 GAC CAA ACT AGC GAA GGG GTT TAC CTT TTC CCT TCT AAA CAG AGG ACG ό " Asp Gin Thr Ser Glu Gly Val Tyr Leu Phe Pro Ser Lys Gin Arg Tnr 210 215 220 Ser Ala Ser T.-.r Met Lei Leu He Lys Ala He Le- He 23C 235 24C AGC TGC ATC AGC AAC AAG TGG CAA CTC ATA TAC TTA CTG TAC TTG ATA 76δ Ser Cys He Ser Asn Lys Trp Gin Leu He Tyr Leu Leu Tyr Leu He 245 250 255 TTT GAA GTG TCT TAC TAC CTC CAC AAG AAA GTT ATA GAT GAA ATA GCT 816 Phe Glu Val Ser Tyr Tyr Leu His Lys Lys Val He Asp Glu He Ala 260 265 270 GGT GGG ACC AAC TTC GTT TCA AGG CTC GTG GCG GCT TTG ATT GAA GTC 864 Gly Gly Thr Asn Phe Val Ser Arg Leu Val Ala Ala Leu He Glu Val 275 280 285 AAT TGG GCC TTC GAC AAT GAA GAA GTC AAA GGC TTA AAG AAG TTC TTC 912 Asn Trp Ala Phe Asp Asn Glu Glu Val Lys Gly Leu Lys Lys Phe Phe 290 295 300 TTG CTG TCT AGT AGG GTC AAA GAG TTG ATC ATC AAA CAC AAA GTG AGG 960 Leu Leu Ser Ser Arg Val Lys Glu Leu He He Lys His Lys Val Arg 305 310 315 320 AAT GAA GTA GTG GTC CGC TGG TTT GGA GAT GAA GAG ATT TAT GGG ATG 1008 Asn Glu Val Val Val Arg Trp Phe Gly Asp Glu Glu He Tyr Gly Met 325 330 335 CCA AAG CTG ATC GGC TTA GTT AAG GCA GCA ACA CTA AGT AGA AAC AAA 1056 Pro Lys Leu He Gly Leu Val Lys Ala Ala Tr.r Leu Ser Arg Asn Lys 340 345 350 CAC TGT ATG TTG TGT ACC GTC TGT GAG GAC AGA GAT TGG AGA GGG GAA 1104 His Cys Met Leu Cys Thr Val Cys Glu Asp Arg Asp Trp Arg Gly Glu 355 360 365 ACT TGC CCT AAA TGT GGG CGT TTT GGA CCA CCA GTG GTC TGC GGT ATG 1152 Thr Cys Pro Lys Cys Gly Arg Phe Gly Pro Pro Val Val Cys Gly Met 370 375 380 ACC CTA GCC GAT TTC GAA GAA AAA CAC TAT AAA AGG ATT TTC ATT AGA 1200 Thr Leu Ala Asp Phe Glu Glu Lys His Tyr Lys Arg He Phe He Arg 385 390 395 400 GAG GAC CAA TCA GGC GGG CCA CTT AGG GAG GAG CAT GCA GGG TAC TTG 1248 Glu Asp Gin Ser Gly Gly Pro Leu Arg Glu Glu His Ala Gly Tyr Leu 405 410 415 CAG TAC AAA GCC AGG GGT CAA CTG TTT TTG AGG AAC CTC CCA GTG TTA 1296 Gin Tyr Lys Ala Arg Gly Gin Leu Phe Leu Arg Asn Leu Pro Val Leu 420 425 430 GCT ACA AAA GTC AAG ATG CTC CTG GTT GGT AAC CTC GGG ACA GAG ATT 1344 Ala Thr Lys Val Lys Met Leu Leu Val Gly Asn Leu Gly Thr Glu He 435 440 445 GGG GAT CTG GAA CAC CTT GGC TGG GTG CTT AGA GGG CCA GCT GTT TGC 1392 Gly Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys 450 455 460 AAG AAG GTT ACT GAA CAC GAA AGA TGC ACC ACG TCT ATA ATG GAT AAG 1440 Lys Lys Val Thr Glu H s Glu Arg Cys Thr Thr Ser He Met Asp Lys 465 470 475 480 TTG ACT GCT TTC TTT GGA GTA ATG CCA AGG GGC ACT ACT CCC AGA GCT 1 88 Leu Thr Ala Phe Phe Gly Val Met Pro Arg Gly Thr Thr Pro Arg Ala 485 490 495 CCC GTA AGA TTC CCT ACC TCC CTC CTA AAG ATA AGA AGA GGG CTG GAG 1 5-. Pro Val Arg Phe Pro Thr Ser Leu Leu Lys He Arg Arg Gly Leu Glu 500 505 510 ACT GGT TGG GCT TAC ACA CAC CAA GGT GGC ATC AGC TCA GTA GAC CAT ". £ Thr Gly Trp Ala Tyr Thr His Gin Gly Gly He Ser Ser Val Asp H s 515 520 525 GTC ACT TGT GGG AAA GAC TTA CTG GTG TGT GAC ACC ATG GGT CGG ACA 1632 Val Thr Cys Gly Lys Asp Leu Leu Val Cys Asp Thr Met Gly Arg Thr 530 535 540 AGG GTT GTT TGC CAG TCA AAT AAT AAG ATG ACC GAC GAG TCC GAA TAC 1680 Arg Val Val Cys Gin Ser Asn Asn Lys Met Thr Asp Glu Ser Glu Tyr 545 550 555 560 GGA GTC AAA ACT GAC TCC GGG TGC CCA GAG GGA GCC AGG TGT TAC GTG 1728 Gly Val Lys Thr Asp Ser Gly Cys Pro Glu Gly Ala Arg Cys Tyr Val 565 570 575 TTT AAC CCG GAA GCA GTT AAC ATA TCA GGC ACT AAA GGA GCC ATG GTC 1776 Phe Asn Pro Glu Ala Val Asn He Ser Gly Thr Lys Gly Ala Met Val 580 585 590 CAC TTA CAG AAA ACG GGT GGA GAA TTC ACC TGT GTG ACA GCA TCA GGA 1824 His Leu Gin Lys Thr Gly Gly Glu Phe Thr Cys Val Thr Ala Ser Gly 595 600 605 ACC CCG GCC TTC TTT GAC CTC AAG AAC CTT AAG GGC TGG TCA GGG CTA 1872 Thr Pro Ala Phe Phe Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu 610 615 620 CCG ATA TTT GAA GCA TCA AGT GGA AGG GTA GTC GGA AGG GTC AAG GTC 1920 Pro He Pne Glu Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val 625 630 635 640 GGG AAG AAC GAG GAT TCC AAA CCA ACC AAG CTC ATG AGT GGG ATA CAA 1968 Gly Lys Asn Glu Asp Ser Lys Pro Thr Lys Leu Met Ser Gly He Gin 645 650 655 ACG GTT TCT AAA AGC GCC ACA GAC TTG ACG GAG ATG GTG AAG AAG ATA 2016 Thr Val Ser Lys Ser Ala Thr Asp Leu Thr Glu Met Val Lys Lys He 660 665 670 ACG ACC ATG AAC AGG GGA GAG TTC AGA CAA ATA ACC CTG GCC ACA GGT 2064 Thr Thr Met Asn Arg Gly Glu Phe Arg Gin He Thr Leu Ala Thr Gly 675 680 685 GCC GGA AAA ACT ACA GAG CTC CCT AGA TCA GTT ATA GAA GAG ATA GGG 2112 Ala Gly Lys Thr Thr Glu Leu Pro Arg Ser Val He Glu Glu He Gly 690 695 700 AGG CAT AAG AGG GTG TTG GTC TTA ATC CCC TTG AGG GCG GCA GCA GAA 2160 Arg His Lys Arg Val Leu Val Leu He Pro Leu Arg Ala Ala Ala Glu 705 710 715 720 TCA GTA TAC CAA TAC ATG AGA CAG AAA CAT CCG AGT ATA GCA TTC AAT 2208 Ser Val Tyr Gin Tyr Met Arg Gin Lys His Pro Ser He Ala Phe Asn 725 730 735 CTA AGG ATA GGT GAG ATG AAG GAA GGT GAT ATG GCC ACG GGA ATA ACC 2256 Leu Arg He Gly Glu Met Lys Glu Gly Asp Met Ala Thr Gly He Thr 740 745 750 TAT GCC TCT TAC GGT TAC TTT TGC CAG ATG TCA CAA CCC AAG CTG AGA 2304 Tyr Ala Ser Tyr Gly Tyr Phe Cys Gin Met Ser Gin Pro Lys Leu Arg 755 760 765 GCC GCA ATG GTA GAA TAT TCC TTT ATA TTC CTA GAT GAG TAT CAT TGT 2352 Ala Ala Met Val Glu Tyr Ser Pne He Pne Leu Asp Glu Tyr His Cys 770 775 780 GCT ACC CCA GAA CAA CTG GCA ATC ATG GGG AAG ATC CAC AGA TTC TCA 2 00 Ala Thr Pro Glu Gin Leu Ala He Met Gly Lys He His Arg Phe Ser 785 790 795 800 GAA AAC CTG CGG GTG GTA GCT ATG ACA GCG ACA CCG GCA GGC ACA GTA 2448 Glu Asn Leu Arg Val Val Ala Met Thr Ala Thr Pro Ala Gly Thr Val 805 810 815 ACA ACC ACT GGG CAG AAA CAC CCT ATA GAG GAA TTT ATA GCC CCG GAA 2496 Thr Thr Thr Gly Gin Lys His Pro He Glu Glu Phe He Ala Pro Glu 820 825 830 GTG ATG AAA GGA GAA GAC TTG GGT TCT GAG TAC TTA GAT ATT GCC GGA 2544 Val Met Lys Gly Glu Asp Leu Gly Ser Glu Tyr Leu Asp He Ala Gly 835 840 845 CTG AAG ATA CCA GTA GAG GAG ATG AAG AAT AAC ATG CTA GTT TTT GTG 2592 Leu Lys He Pro Val Glu Glu Met Lys Asn Asn Met Leu Val Phe Val 850 855 e60 CCC ACC AGG AAC ATG GCG GTA GAG GCG GCA AAG AAA TTG AAG GCC AAA 2640 Pro Thr Arg Asn Met Ala Val Glu Ala Ala Lys Lys Leu Lys Ala Lys 865 870 875 880 GGA TAC AAC TCG GGC TAC TAC TAC AGC GGA GAG GAC CCA TCT AAC CTG 2688 Gly Tyr Asn Ser Gly Tyr Tyr Tyr Ser Gly Glu Asp Pro Ser Asn Leu 885 δ90 895 AGG GTG GTG ACG TCG CAG TCC CCA TAC GTG GTG GTA GCA ACC AAC GCA 2736 Arg Val Val Thr Ser Gin Ser Pro Tyr Val Val Val Ala Thr Asn Ala 900 905 910 ATA GAA TCG GGC GTT ACC CTC CCG GAC CTG GAC GTG GTT GTC GAC ACG 2784 He Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Val Val Val Asp Thr 915 920 925 GGA CTC AAG TGT GAA AAA AGA ATC CGA CTG TCA CCC AAG ATG CCT TTC 2832 Gly Leu Lys Cys Glu Lys Arg He Arg Leu Ser Pro Lys Met Pro Phe 930 935 940 ATA GTG ACG GGC CTG AAA AGA ATG GCC GTC ACT ATT GGG GAA CAA GCC 2880 He Val Tnr Gly Leu Lys Arg Met Ala Val Thr He Gly Glu Gin Ala 945 950 955 960 CAG AGA AGA GGG AGG GTT GGA AGA GTG AAG CCC GGG AGA TAC TAC AGG 2928 Gin Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr Tyr Arg 965 970 975 AGT CAA GAA ACA CCT GTC GGC TCT AAA GAC TAC CAT TAT GAC TTA TTG 2976 Ser Gin Glu Thr Pro Val Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu 980 985 990 CAA GCC CAG AGG TAC GGC ATA GAA GAT GGG ATA AAT ATC ACC AAA TCC 3024 Gin Ala Gin Arg Tyr Gly He Glu Asp Gly He Asn He Thr Lys Ser 995 1000 1005 TTC AGA GAG ATG AAC TAC GAC TGG AGC CTT TAT GAG GAA GAT AGC CTG 3072 Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu Tyr Glu Glu Asp Ser Leu 1010 1015 1020 ATG ATC ACA CAA CTG GAA ATC CTC AAC AAC CTG TTG ATA TCA GAA GAG 3120 Met He Thr Gin Leu Glu He Leu Asn Asn Leu Leu He Ser Glu Glu 1025 1030 1035 1040 CTG CCG ATG GCA GTA AAA AAT ATA ATG GCC AGG ACC GAC CAC CCA GAA 3168 Leu Pro Met Ala Val Lys Asn He Met Ala Arg Thr Asp His Pro Glu 1045 1050 1055 CCA ATT CAA CTC GCG TAT AAC AGC TAC GAG ACA CAG GTG CCG GTA TTA 2216 Pro He Gin Leu Ala Tyr Asn Ser Tyr Glu Thr Gin Val Pro Val Leu 1060 1065 1070 TTC CCA AAA ATA AGA AAT GGA GAG GTG ACT GAT ACT TAC GAT AAT TAC 3264 Phe Pro Lys He Arg Asn Gly Glu Val Thr Asp Thr Tyr Asp Asn Tyr 1075 1080 1085 ACC TTC CTC AAT GCA AGA AAA TTG GGA GAT GAC GTA CCC CCC TAC GTG 2312 Thr Phe Leu Asn Ala Ar Lys Leu Gly Asp Asp Val Pro Pro Tyr Val 1090 1095 HOG TAT GCT ACA GAG GAT GAG GAC TTG GCA GTG GAA CTG TTG GGC CTA GAT Ξ360 Tyr Ala Thr Glu Asp Glu Asp Leu Ala Val Glu Leu Leu Gly Leu Asp 1105 1110 1115 112C TGG CCG GAC CCA GGA AAC CAA GGC ACC GTG GAA GCT GGC AGA GCA CTA 3408 Trp Pro Asp Pro Gly Asn Gin Gly Thr Val Glu Ala Gly Arg Ala Leu 1125 1130 1135 5 AAA CAG GTG GTT GGT CTA TCA ACA GCA GAG AAC GCC CTG CTA GTC GCC 3456 Lys Gin Val Val Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Val Ala 1140 1145 1150 CTG TTC GGC TAC GTG GGG TAC CAG GCG CTT TCA AAG AGA CAT ATA CCA 3504 0 Leu Phe Gly Tyr Val Gly Tyr Gin Ala Leu Ser Lys Arg His He Pro 1155 1160 1165 CTG GTC ACA GAT ATA TAT TCA GTA GAA GAT CAC AGG CTA GAG GAC ACT 3552 Leu Val Thr Asp He Tyr Ser Val Glu Asp His Arg Leu Glu Asp Thr 5 1170 1175 1180 ACG CAC CTA CAG TAT GCT CCG AAT GCC ATC AAG ACG GAG GGG AAG GAA 3600 Thr H s Leu Gin Tyr Ala Pro Asn Ala He Lys Thr Glu Gly Lys Glu 1185 1190 1195 1200 ACT GAA TTG AAG GAG CTG GCT CAG GGG GAT GTG CAG AGA 3639 Thr Glu Leu Lys Glu Leu Ala Gin Gly Asp Val Gin Arg 1205 1210 * ι INFORMATION FOR SE2 ID NO (ι) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1213 amino acids 0 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 5 Lys Gly Gly Lys He Asp Gly Gly Trp Gin Arg Gin Pre Val Thr Ser 1 5 10 15 Phe Asp He Gin Leu Ala Leu Ala Val Val Val Val Val Val Met Leu 20 25 30 Leu Ala Lys Arg Asp Pro Thr Thr Phe Pro Leu Val He Thr Val Ala 35 40 45 Thr Leu Arg Thr Ala Lys He Thr Asn Gly Phe Ser Thr Asp Leu Val 50 55 60 He Ala Thr Val Ser Ala Ala Leu Leu Thr Trp Thr Tyr He Ser Asp 65 70 75 80 Tyr Tyr Lys Tyr Lys Thr Trp Leu Gin Tyr Leu Val Ser Thr Val Thr Θ5 90 95 Gly He Pne Leu He Arg Val Leu Lys Gly He Gly Glu Leu Asp Leu 100 105 110 His Ala Pro Thr Leu Pro Ser H s Arg Pro Leu Phe Tyr He Leu Val 115 120 125 Tyr Leu He Ser Thr Ala Val Val Thr Arg Trp Asn Leu Asp Val Ala 130 135 140 Gly Leu Leu Leu G_r. Cys Val Pro Thr Leu Leu Met Val Pne Tr.r Met 145 150 155 160 Trp Ala Asp He Leu Thr Leu He Leu He Leu Pro Thr Tyr Glu Leu 165 170 175 Thr Lys Leu Tyr Tyr Leu Lys Glu Val Lys He Gly Ala Glu Arα Gly 180 185 190 Trp Leu Trc Lys Tr.r Asn Tyr Lys Arg Val Asn Asp lie Tyr Glu Val 195 200 205 Asp Gin Thr Ser Glu Gly Val Tyr Leu Pne Pro Ser Lys Gin Arg Thr 210 215 220 Ser Ala He Thr Ser Thr Met Leu Pro Leu He Lys Ala He Leu He 225 230 235 240 Ser Cys He Ser Asn Lys Trp Gin Leu He Tyr Leu Leu Tyr Leu He 245 250 255 Phe Glu Val Ser Tyr Tyr Leu His Lys Lys Val He Asp Glu He Ala 260 265 270 Gly Gly Thr Asn Phe Val Ser Arg Leu Val Ala Ala Leu He Glu Val 275 280 285 Asn Trp Ala Phe Asp Asn Glu Glu Val Lys Gly Leu Lys Lys Phe Phe 290 295 300 Leu Leu Ser Ser Arg Val Lys Glu Leu He He Lys His Lys Val Arg 305 310 315 320 Asn Glu Val Val Val Arg Trp Phe Gly Asp Glu Glu He Tyr Gly Met 325 330 335 Pro Lys Leu He Gly Leu Val Lys Ala Ala Thr Leu Ser Arg Asn Lys 340 345 350 His Cys Met Leu Cys Thr Val Cys Glu Asp Arg Asp Trp Arg Gly Glu 355 360 365 Thr Cys Pro Lys Cys Gly Arg Phe Gly Pro Pro Val Val Cys Gly Met 370 375 380 Thr Leu Ala Asp Phe Glu Glu Lys His Tyr Lys Arg He Phe He Arg 385 390 395 400 Glu Asp Gin Ser Gly Gly Pro Leu Arg Glu Glu His Gly Tyr Leu 405 410 415 Gin Tyr Lys A.la Arg Gly Gin Leu Phe Leu Arg Asn Leu Pro Val Leu 420 425 430 Ala Thr Lys Val Lys Met Leu Leu Val Gly Asn Leu Gly Thr Glu He 435 440 445 Gly Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys 450 455 460 Lys Lys Val Thr Glu His Glu Arg Cys Thr Thr Ser He Met Asp Lys 465 470 475 480 Leu Thr Ala Phe Phe Gly Val Met Pro Arg Gly Thr Thr Pro Arg Ala 485 490 495 Pro Val Arg Phe Pro Thr Ser Leu Leu Lys He Arg Arg Gly Leu Glu 500 505 510 Thr Gly Trp Ala Tyr Thr His Gin Gly Gly He Ser Ser Val Asp His 515 520 525 Val Thr Cys Gly Lys Asp Leu Leu Val Cys Asp Thr Met Gly Arg Thr 530 535 540 Arg Val Val Cys Gin Ser Asn Asn Lys Met Thr Asp Glu Ser Glu Tyr 545 550 555 560 Gly Val Lys Thr Asp Ser Gly Cys Pro Glu Gly Ala Arg Cys Tyr Val 565 570 575 Phe Asn Pro Glu Ala Val Asn He Ser Gly Thr Lys Gly Ala Met Val 580 585 590 His Leu Gin Lys Thr Gly Gly Glu Phe Thr Cys Val Thr Ala Ser Gly 595 600 605 Thr Pro Ala Phe Phe Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu 610 615 620 Pro He Phe Glu Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val 625 630 635 640 Gly Lys A.sn Glu Asp Ser Lys Pro Thr Lys Leu Met Ser Gly He Gin 645 650 655 Thr Val Ser Lys Ser Ala Thr Asp Leu Thr Glu Met Val Lys Lys He 660 665 670 Thr Thr Met Asn Arg Gly Glu Phe Arg Gin He Thr Leu Ala Thr Gly 675 680 685 Ala Gly Lys Thr Thr Glu Leu Pro Arg Ser Val He Glu Glu He Gly 690 695 700 Arg His Lys Arg Val Leu Val Leu He Pro Leu Arg Ala Ala Ala Glu 705 710 715 720 Ser Val Tyr Gin Tyr Met Arg Gin Lys H s Pro Ser He Ala Phe Asn 725 730 735 Leu Arg He Gly Glu Met Lys Glu Gly Asp Met Ala Thr Gly He Thr 740 745 750 Tyr Ala Ser Tyr Gly Tyr Phe Cys Gin Met Ser Gin Pro Lys Leu Arg 755 760 765 Ala Ala Met Val Glu Tyr Ser Pne He Phe Leu Asp Glu Tyr His Cys 77C ^ 75 780 Aia Thr Pro Glu Gin Leu Ala He Met Gly Lys He His Arg Phe Ser 785 790 795 800 Glu Asn Leu Arg Val Val Ala Met Thr Ala Thr Pro Ala Gly Thr Val 805 810 815 Thr Tnr Thr Gly Gin Lys His Pre He Glu Glu Pne He Ala Pre Glu 820 825 830 Val Met Lys Gly Glu Asp Leu Gly Ser Glu Tyr Leu Asp He Aia Gly 835 840 845 Leu Lys He Pro Val Glu Glu Met Lys Asn Asn Met Leu Val Phe Val 850 855 860 Pro Thr Arg Asn Met Ala Val Glu Ala Ala Lys Lys Leu Lys Ala Lys 865 870 875 880 Gly Tyr Asn Ser Gly Tyr Tyr Tyr Ser Gly Glu Asp Pro Ser Asn Leu 885 890 895 Arg Val Val Thr Ser Gin Ser Pro Tyr Val Val Val Ala Thr Asn Ala 900 905 910 He Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Val Val Val Asp Thr 915 920 925 Gly Leu Lys Cys Glu Lys Arg He Arg Leu Ser Pro Lys Met Pro Phe 930 935 940 He Val Thr Gly Leu Lys Arg Met Ala Val Thr He Gly Glu Gin Ala 945 950 955 960 Gin Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr Tyr Arg 965 970 975 Ser Gin Glu Thr Pro Val Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu 980 9δ5 990 Gin A_a Gin A.rg Tyr Gly He Glu Asp Gly He Asn lie Thr Lys Ser 995 1000 1005 Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu Tyr Glu Glu Asp Ser Leu 1010 1015 1020 Met He Thr Gin Leu Glu He Leu Asn Asn Leu Leu He Ser Glu Glu 1025 1030 1035 1040 Leu Pro Met Ala Val Lys Asn He Met Ala Arg Thr Asp H s Pro Glu 1045 1050 1055 Pro He Gin Leu Ala Tyr Asn Ser Tyr Glu Thr Gin Val Pro Val Leu 1060 1065 1070 Phe Pro Lys He Arg Asn Gly Glu Val Thr Asp Thr Tyr Asp Asn Tyr 1075 lθeθ 1085 Thr Phe Leu Asn Ala Arg Lys Leu Gly Asp Asp Val Pro Pro Tyr Val 1090 1095 1100 Tyr Ala Thr Glu Asp Glu Asp Leu Ala Val Glu Leu Leu Gly Leu Asp 1105 1110 1115 1120 Trp Pro Asp Pro Gly Asn Gin Gly Thr Val Glu Ala Gly Arg Ala Leu 1125 1130 1135 Lys Gin Val Val Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Val Ala 1140 1145 1150 Leu Phe Gly Tyr Val Gly Tyr Gin Ala Leu Ser Lys Arg His He Pro 1155 1160 1165 Leu Val Thr Asp He Tyr Ser Val Glu Asp His Arg Leu Glu Asp Thr 1170 1175 1180 Thr His Leu Glr. Tyr Aia Pro Asn Ala He Lys Thr Glu Gly Lys Glu 1185 1190 1195 1200 Thr Glu Leu Lys Glu Leu Ala Gin Gly Asp Val Gin Arg 1205 1210 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D; TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Classical Swine Fever Virus (B) STRAIN: Alfort (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: I..45 (χι) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TCA ACA GCA GAG AAC GCC CTG CTA GTC GCC CTG TTC GGC TAC GTG 45 Ser Thr Ala Glu Asn Ala Leu Leu Val Ala Leu Phe Gly Tyr Val 1215 1220 1225 (2) INFORMATION FOR SEQ ID NO: 4: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 ammo acids ( , TYPE: ammo acid (D TOPOLOGY: linear (11) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Ser Thr Ala Glu Asn Ala Leu Leu Val Ala Leu Phe Gly Tyr Val 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 5: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (IV) ANTI-SENSE: NO (VI) ORIGINAL SOURCE: (A) ORGANISM: Claasical Swine Fever Virus (B) STRAIN: Alfort (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:!..27 (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GAG AAC GCC CTG CTA GTC GCC CTG TTC 27 Glu Asn Ala Leu Leu Val Ala Leu Phe 20 (2) INFORMATION FOR SEQ ID NO: 6: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 am o acids (B) TYPE: ammo acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (XI) SEQUENCE DESCRIPTION: SEQ ID NO: c Glu Asn Ala Leu Leu Val Ala Leu Phe 1 5

Claims: CLAIMS 1. A vaccine for the protection of swine against classical swine fever (CSF) , characterized m that it comprises CSF virus polypeptide plO or an lmmunogenically active part thereof, together with a pharmaceutically acceptable carrier. 2. A vaccine according claim 1, characterized in that the plO polypeptide has an ammo acid sequence shown in SEQ ID NO: 2. 3. A vaccine for the protection of swme against CSF, characterized in that it comprises a recombinant micro¬ organism harbouring a DNA sequence encoding the polypeptide defined in claim 1 or 2, together with a pharmaceutically acceptable carrier. 4. A vaccine according to claim 3, characterized in that the recombinant micro-organism is a recombinant vector virus, preferably recombinant pseudorabies virus.

Inventors: ELBERS KNUT, PAULY THOMAS, THIEL HEINZ-JURGEN, ELBERS, KNUT, PAULY, THOMAS, THIEL, HEINZ-JURGEN
Applicants: AKZO NOBEL NV, ELBERS KNUT, PAULY THOMAS, THIEL HEINZ JURGEN, AKZO NOBEL N.V., ELBERS, KNUT, PAULY, THOMAS, THIEL, HEINZ-JURGEN
Application year: 1995
Main classification: C07K14
IPC codes: A61K39, A61P31, C07K14, C12N5, C12N15, C12P21, C12R1