CN1307638A

CN1307638A - Host-encoded protein expressed on Marek's disease (MDV)-infected cells and antibody thereto

Info

Publication number: CN1307638A
Application number: CN99808080A
Authority: CN
Inventors: S·C·布格斯; T·F·达维森; L·J·N·罗斯
Original assignee: INST FOR ANIMAL HEALTH Ltd
Current assignee: INST FOR ANIMAL HEALTH Ltd
Priority date: 1998-04-29
Filing date: 1999-03-22
Publication date: 2001-08-08
Also published as: WO1999055860A1; GB9809070D0; EP1073735A1; NO20005321L; NO20005321D0; AU3618099A; CA2327178A1; WO1999055860A8; JP2002518995A

Abstract

The invention relates to nucleic and amino acid sequences which encode an antigen which is associated with avian tumours, particularly avian tumours associated with Marek's disease virus. The invention also relates to the use of the sequences in medicine, particularly in the production of compositions useful in the treatment and prophylaxis of avian tumours.

Description

The host-encoded protein and the antibody thereof of expressing on horsepower creutzfeldt jakob disease (MDV)-cells infected

The present invention relates to encode with tumour particularly the fowl tumour especially with oncovirus such as relevant relevant antigenic nucleic acid and the aminoacid sequence of fowl tumour of marek disease disease virus.The invention still further relates to the expression vector of having integrated above-mentioned sequence and with these carriers and antigen at medicine, the purposes of tumor treatment and prevention aspect particularly.

Marek disease disease virus (MDV) is the relevant fowl α simplexvirus of a kind of height cell, it can produce a kind of lymphocytic hyperplasia disease, promptly horsepower creutzfeldt jakob disease (MD), taking the form of of this disease produces DPN and/or lymphoma [Payne in the viscera tissue of susceptible bird, L.N. summarize (editor), " horsepower creutzfeldt jakob disease " (1985), Martinus Nijhoff Publishing, Boston].MD has important economic implications to global fowl industry.The bacterial strain of MDV is divided into 3 serotypes.1 C-type virus C comprises carcinogenic bacterial strain and their attenuated strain, and the pathotype of 1 different C-type virus Cs has nothing in common with each other, from only produce the gentle carcinogenic C-type virus C of MD the susceptible bird, up to the high carcinogenic type (high toxicity+) that can produce MD in the bird of immunity.2 type MDV are infective but not carcinogenic natural bacterial strains.Serotype 3 C-type virus Cs comprise closely-related herpes turkey virus (HVT), and this virus does not all have pathogenic in turkey and chicken.At present, can obtain from commercial channels based on the vaccine of all three kinds of serotype viruses, they can be used in combination separately or by different way.Nearly all poultry that is used for commercial production has all inoculated vaccine, but continue to have the report of clinical disease outburst, and incidence and seriousness are all improving, because [Witter, R.L. (1997) " bird research " 41:149-163] constantly appears in the high toxicity pathotype of MDV.In the area of the outburst especially severe of MD, the solution that the poultry producer adopts is then uses and have more the vaccine of invasiveness, and seeks help from and use combined vaccine (two valencys or trivalent) to improve protectiveness.But production of vaccine person is accepting a kind of like this viewpoint, promptly uses traditional MD vaccine can't control MD constantly.

The present invention attempts to provide evaluation, prevention or treatment useful method and the reagent to tumour.

Aspect first, the invention provides a kind of polypeptide of purifying, this polypeptide can be discerned by hybridoma AV37 excretory monoclonal antibody, wherein this hybridoma was deposited in European zooblast preservation center (ECACC), Centre forApplied Microbiology according to budapest treaty on March 3rd, 1998; Research, Salisbury, Wiltshire SP4OJG, UK, preserving number are 98030304.

Be used for herein, the meaning of " purifying " is, polypeptide does not contain its some biological substance when nature is found, preferably most biological substances, and in other words, polypeptide does not exist with the existence form before its.Therefore, polypeptide of the present invention comprises that such as the polypeptide in a kind of composition, said composition contains a kind of pharmaceutically acceptable supporting agent or vehicle or a kind of adjuvant.

Polypeptide preferably has the AV37 polypeptide antigen of the sequence that is shown in Fig. 5 (SEQ ID NO.2) and the varient and the fragment of this polypeptide.

" fragment " and " varient " be meant those can be used for preparing can specificity in conjunction with the antibody of said polypeptide or its mutant forms but lack the polypeptide of the function of natural polypeptides.This varient and fragment generally include at least one zone of containing at least 5 continuous amino acids, and 5 of the homologous of this zone and said polypeptide or more amino acid region have at least 90% homology.Fragment is less than 100% whole polypeptide.For example, as a fragment of the AV37 polypeptide that is shown in Fig. 5 (SEQ ID NO.2), amino acid can be prepared into a sequence NH with AV37 polypeptide ₂The synthetic polypeptide of-CDTLKNWFYDETLGRC-COOH (SEQ ID NO.3).It is believed that this sequence is positioned at the outside of protein structure, thereby can excite a kind of antibody response.

The amino acid that is shown in Fig. 5 (SEQ ID NO.2) also can be prepared into the sequence NH that has the AV37 polypeptide respectively respectively ₂-DVMVPVEEEGKEFHHPTTATEK-COOH (SEQ ID NO.4) (C-terminal peptide) and NH ₂The synthetic peptide of-QPPFTSSHSCDTLKNWFYDETL-COOH (SEQ ID NO.5) (N-terminal peptide).The sequence of back is from C and N-terminal, and these peptides are known to be outstanding candidate's peptide of anti-this peptide antibody of preparation, promptly as peptide vaccine, because they have better movability (flexibility) than the peptide that is fixed in the protein three-dimensional structure.More the result of high flexibility is, some of peptide formed the easier native conformation of taking of unit, thereby makes the antibody that produces at them more can discern natural antigen.

It will be understood by those of skill in the art that polypeptide of the present invention can modify by known peptide modified technology.These technology comprise disclosed technology in the US Patent No 4,302,386 of authorizing Stevens on November 24th, 1981, and this patent is hereby incorporated by.This modification can increase immunogenicity of antigens, and perhaps they can be to the not influence of such immunogenicity.For example, a few amino acids can be changed.In addition, antigen of the present invention can contain one or more to the optional aminoacid sequence of its immunogenicity.Undesired sequence can be removed by technology well known in the art.For example, these sequences can be used enzyme such as casease or papoid or proteins associated lytic enzyme to digest by limited proteolysis to remove.

In addition, being equivalent to the little polypeptide of the antigenic portions of polypeptide can be by method chemosynthesis well known in the art.This comprises disclosed method in the US Patent No 4,290,944 of authorizing Goldberg on December 22nd, 1981, and this patent is hereby incorporated by.

Therefore, polypeptide of the present invention comprises a class modified polypeptides, comprises the polypeptide in synthetic source or the fragment of original polypeptide, and they all have initial, structure and immunogenic mutual component, and all within the scope of the invention.

Polypeptide also can synthesize by solid-phase peptide synthetic Fmoc-polymeric amide pattern, as disclosing of (1981) " J.Org.Chem " 46:3433 such as Lu and reference thereof.The temporary protection of N-amino group is provided by 9-fluorenylmethyloxycarbonyl (Fmoc) group.The piperidines that repeats to cut use 20% in N, dinethylformamide of this high-alkali unstable blocking group carries out.Functional side chain can their butyl ester (when Serine, Threonine and tyrosine), butyl ester (when L-glutamic acid and aspartic acid), butyl carbonyl derivative (when Methionin and Histidine), trimethylphenyl derivative (during halfcystine) and 4-methoxy-2,3,6-trimethylbenzene sulfone derivatives (during arginine) form be protected.When glutamine or l-asparagine are the C-terminal residue, use 4,4 '-veratryl radical protection side chain amino functional.The solid phase support is based on the polydimethylacrylamiin polymer, and the latter is by three kinds of monomers--and-DMAA (skeleton monomer), bisacrylamide diamines (linking agent) and propenyl sarkosine methyl esters (function reagent) are formed.It is sour unsettled 4-p hydroxymethylphenoxyacetic acid derivative that the peptide that uses-resin connects reagent.All amino acid derivative all add with the form of its homotype anhydride ester derivs, and except glutamine or l-asparagine, they use a kind of reversible N, the coupling method of N-dicyclohexylcarbodiimide/I-hydroxybenzotriazole mediation.All couplings and deprotection reaction all use triketohydrindene hydrate, trinitro-benzene-sulfonic acid or isotope detection test to monitor.Behind the end of synthesis, handle, peptide is downcut from the resin upholder, remove the side chain protected group simultaneously with 95% trifluoroacetic acid that contains 50% scavenging agent.Normally used scavenging agent is ethylene glycol, phenol, methyl-phenoxide and water.The accurate amino acid of selecting to depend on institute's synthetic peptide is formed.Trifluoroacetic acid is removed by vacuum volatilization, with the diethyl ester development, provides rough peptide subsequently.The scavenging agent of any existence is all removed by a simple extractive process, and this process provides the first system peptide that does not contain scavenging agent by the liquid phase freeze-drying.Peptide synthetic reagent is generally from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, and UK obtains.Purifying can be undertaken by making up such as any of molecular size exclusion chromatography, ion-exchange chromatography and (mainly) RPLC technology or its.The analysis of peptide can be used the amino acid analysis after thin-layer chromatography, RPLC, the acid hydrolysis and be undertaken by high speed atom bombardment (FAB) mass spectroscopy.

Can use method well known in the art to identify with AV37 monoclonal antibody bonded appropriate peptide part of the present invention.

A kind of method is open by (1990) " PNAS " 87:6378-6382 such as Scott and Smith (1990) " science " 249:386-390 and Cwirla, it relates to the big library screening of filobactivirus such as M13 or fd, the peptide that each member in this library has the different albumen with on the phage surface to merge.Can use a kind of combining method repeatedly to screen with AV37 monoclonal antibody bonded library member, in case purifying the phage that can combine closely most, the sequence of peptide part can check order by the DNA to the coded surface fusion rotein simply and measure.Another kind of operable method is NovaTope (TM) system, and this system can be from Novagen, Inc., and 597 Science Drive, Madison, WI53711 buys.The basis of this method is the library of preparation bacterial clone, and each bacterial clone all stably express has been considered to part and has existed from a kind of little peptide of candidate albumen in the said candidate albumen.Screen with the rendering method of standard in the library, uses antibody or other binding reagents as probe.Positive colony can directly be measured the accurate aminoacid sequence of part by the dna sequencing analysis.

Further method also can be used to identify the peptide part, as the matrix of the single synthetic peptide sequence on the disclosed solid support in United States Patent (USP) 5143854 such as disclosed synthetic peptide combinatorial library of (1991) " nature " 354:84-86 such as the peptide library that uses the single kind of the disclosed microballon bonded of (1991) " nature " 354:82-84 such as Lam, Houghten or Pirrung.

Can use the monoclonal antibody technique preparation of standard in conjunction with the monoclonal antibody of AV37 polypeptide and its fragment and varient.Antigen-binding portion thereof can be an a kind of part (as the Fab fragment) or a kind of synthetic antibody fragment (as a kind of strand Fv fragment [ScFv]) of antibody.Be used to select antigenic suitable monoclonal antibody to prepare by known technology, for example in " monoclonal antibody: technical manual ", H Zola (CRC Press, 1988) and " mono-clonal hybridoma antibody: technology and application ", disclosed technology among the J G R Hurrel (CRC Press, 1982).

Chimeric antibody is by (1988, the 8 international biotechnology conferences, 2 parts, 792-799) discussion such as Neuberger.

One or more amino-acid residues are before peptide is synthetic or synthesize the function that afterwards be can be used to provide this peptide by the peptide of chemically modified, and the ability that promptly produces specific antibody in vivo remains unchanged basically.This modification comprises and acid or alkali formation salt, especially with physiologically-acceptable organic or inorganic acid or alkali, forms the ester or the acid amides of terminal carboxyl groups, and in conjunction with upper amino acid blocking group such as N-tertbutyloxycarbonyl.This modification can be protected not metabolism in vivo of peptide.Peptide can singly copy or multiple copied exists as series connection multiple form.This series connection or multiple copied repeat to make them have effective antigens, and avoid using a kind of carrier.It is that N-terminal and C-terminal link together or terminal add one or more halfcystines to increase antigenicity and/or to form disulfide linkage at one that peptide is preferably formed a kind of ring.If peptide combines with preferably a kind of polypeptid covalence of a kind of carrier, its arrangement mode preferably makes peptide of the present invention form a ring.

According to current immunology theory, the function of carrier should be the stimulation that is present in any immunogenic formulation with stimulating immune system or enhancing immunity system.It is believed that optimum carrier contains (or produce with antigen) a kind of t cell epitope.Peptide can with a kind of independent carrier such as serum albumin, myohaemoglobin, bacterial toxoid and keyhole azurin by linking together such as cross-linking method.Grow up in the time of more recently can induce immune response the auxiliary carrier of T cell comprise the 163-171 peptide of hepatitis B B cAg (being also referred to as nucleocapsid protein), the t cell epitope of inferring such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase enzymes and Bai Jie-1.Back one compound can be considered to a kind of carrier or a kind of adjuvant or both.In addition, the several copies with a kind of or different polypeptide of the present invention can be cross-linked with each other, and just do not have independent carrier in this case, but the function of carrier can be by this crosslinked providing.Suitable cross-linking reagent comprises the reagent of listing in Sigma and Pierce catalogue, glutaraldehyde, carbodiimide and succinimide-4-(N-maleimide methyl) hexanaphthene-1-carboxylate for example, back one reagent utilize C-terminal cysteine residues-SH group (if present).

If polypeptide is to prepare by express a kind of suitable nucleotide sequence in a kind of suitable host, that just preferred this peptide of formal representation with fusion product is promptly with a kind of peptide sequence amalgamation and expression as carrier." Ecosec " of Kabigen is an example of this arrangement.

Aspect second, the present invention relates to the purposes of polypeptide of the present invention in the preparation medicinal composition.Specifically, be used to produce vaccine, this vaccine is used for the treatment of or prophylaxis of tumours, and preferably the bird tumour is especially viral as MDV, ALV and the relevant bird tumour of reticuloendothelial cell virus of proliferation with carcinogenic bird.

Preferably carry out the active immunity of acceptor.In this mode, one or more AV37 polypeptide/albumen prepare in a kind of immunogenic formulation that contains suitable adjuvant and carrier, and deliver medicine to acceptor by known approach.In bird, injection is a kind of mode of administration easily in the ovum of zygote.Suitable adjuvant comprises that Fu Shi fully or " Iscoms ", aluminium hydroxide, saponin(e, DEAE-dextran, neutral oil (for example miglyol), vegetables oil (as peanut oil), liposome, Pluronic polyvalent alcohol or the Ribi adjuvant system (being seen in as GB-A-2 189 141) of Freund, Muramyl dipeptide, EP109 942, EP 180 564 and EP 231 039." Pluronic " is a kind of registered trademark.

For better immune effect is provided, the preferred use be not the AV37 polypeptide for preparing in the species that will handle.Another kind of polypeptide can be used to replace the full molecule of AV37 polypeptide, produces inhibiting antibody in patient.Other polypeptide like this comprise proteic fragment of AV37 and analogue.Such polypeptide can screen according to top introduction, guarantees that they can produce inhibiting antibody in acceptor.

The 3rd aspect of the present invention provides a kind of Fig. 5 of being shown in the separation AV37 gene and the varient thereof of (SEQ ID No.1):

Be used for herein, the meaning of " separation " speech is, this gene separates from its found genomic major part at least, and in other words, this gene is not to exist with the form that exists before its.Therefore, for example, gene of the present invention comprises such gene, and it is advanced a kind of bacteria carrier for example plasmid or a kind of virus vector such as phage by the clone, as long as such clone separates from the clone who is made up of the DNA library of related gene group.

" gene " can comprise its expression promoter of common control and/or other expression-adjusting sequence, and it can comprise intron, and perhaps it can only be made up of encoding sequence, for example the cDNA sequence.

" varient " of gene is such material, and (i) it can be used to prepare a peptide species or an one fragment, and the latter can be used to again prepare can be specifically in conjunction with the proteic antibody by said genes encoding; Or (ii) corresponding to the antisense sequences of this gene or a kind of (i) defined above form variation body.For example, can replace with codon different with original codon but the coding same amino acid.In addition, replace a kind of different amino acid of codon codified, but do not influence proteic immunocompetence, or improve its activity or immunogenicity.For example, site-directed mutagenesis or other technologies can be used to produce single or multiple mutation, for example replace, insertion, deletion and transposition, the introduction in " strategy of vitro mutagenesis and application " " science " 229:193-1210 (1985) as Bostein and Shortle, this article is hereby incorporated by.Because such modifying factor can be used the technology of knowing according to the introduction here and obtain, such modifying factor is also contained in the scope of the present invention.

And it will be understood by those of skill in the art that gene order of the present invention (or its fragment) can be used for obtaining can be under highly rigorous condition and other dna sequence dnas of its hybridization.This DNA comprises any genomic dna.Therefore, gene of the present invention comprise show with have 55% by method genes identified of the present invention, the DNA of preferred 60%, most preferably 70% homology, as long as the albumen of a kind of method that can be used for introducing below of this homologous dna coding.In addition, the identity at least about 35% is arranged between aminoacid sequence that the homologous gene in other species can be by proof prediction and the aminoacid sequence in the obtainable albumen database, preferred 35,40,50,60 or bigger identity, identify.

DNA-DNA, DNA-RNA and RNA-RNA hybridization can be in containing the liquor of 0.1XSSC to 6XSSC, carry out under 55 ℃ to 65 ℃ temperature.This area is known, and temperature is high more, and SSC concentration is low more, and hybridization conditions is just rigorous more." highly rigorous " is used to refer to 2XSSC and 65 ℃.1XSSC is a 0.15M NaCl/0.015M Trisodium Citrate.

" varient " of gene comprises such gene, (for example 20 to 50 Nucleotide) have the homology (at least 50% of height with the allele of gene of the present invention on the less relatively fragment of this gene, preferably at least 90 or 95%), even the whole homology of two kinds of genes may be very low.This is because even proteic general structure difference, but important activity or binding site may be identical.

The present invention comprises that also the predicted amino acid sequence that shows with genes encoding of the present invention has at least 30% identity, preferred 35,40,50,60 or the aminoacid sequence of the identity of bigger %.

Dna sequence dna of the present invention is preferably expressed in a kind of appropriate host and is prepared polypeptide of the present invention.Therefore, the DNA of the polypeptide of the present invention of encoding can make amendment according to known technology and according to the introduction here, is used for construction of expression vector, and the latter can be used to again transform a kind of proper host cell, is used for expressing and preparing polypeptide of the present invention.This technology comprises the U.S. Patent No. 4 of authorizing Rutter etc. on March 3rd, 1984,440,859, authorized the U.S. Patent No. 4 of Weissman etc. on July 23rd, 1985,530,901, authorized the U.S. Patent No. 4 of Crowl etc. on April 15th, 1986,582,800, authorized the U.S. Patent No. 4,677,063 of Mark etc. on June 30th, 1987, authorized the U.S. Patent No. 4 of Goeddel etc. on July 7th, 1987,678,751, authorized the U.S. Patent No. 4,704,362 of Itakura etc. on November 3rd, 1987, authorized the U.S. Patent No. 4 of Murray etc. on December 1st, 1987,710,463, authorized Toole, the U.S. Patent No. 4,757 of Jr. etc. on July 2nd, 1988,006, authorized the U.S. Patent No. 4 of Goeddel etc. on August 23rd, 1988, authorized the U.S. Patent No. 4,810 of Stalker etc. on March 7th, 776,075 and 1989, disclosed technology in 648, above-mentioned patent is hereby incorporated by.

The DNA of polypeptide of the present invention of encoding can be connected to form carrier with multiple other dna sequence dnas, is used for importing appropriate host.Whether essence, DNA that companion DNA depends on the host import host's mode and need episome to keep or integrate.

DNA generally is inserted into a kind of expression vector with suitable direction and correct reading frame and for example is used for expressing in the plasmid.If desired, DNA can discern with required host, and suitable transcribing with translating regulated the control nucleotide sequence and is connected, even this controlling elements generally can be by obtaining in the expression vector.By standard technique carrier is imported the host then.In general, all suppressed by vector conversions of not every host.Therefore, need select transformed host cells.A kind of select technology relate to in transformed host cells the coding a kind of selective marker for example dna sequence dna and any essential controlling elements of antibiotics resistance are incorporated in the expression vector.In addition, the gene of this selective marker of encoding can be on another kind of carrier, and this carrier is used to the required host cell of cotransformation.

Then recombinant DNA transformed host cells those skilled in the art of the present invention is known, cultivate time enough down according to the conditions suitable of disclosure herein content introduction, make expression of polypeptides, reclaim polypeptide then.

Multiple expression system is known, comprises bacterium (for example intestinal bacteria and subtilis), yeast (for example cereuisiae fermentum), filamentous fungus (for example aspergillus), vegetable cell, zooblast and insect cell.

Carrier comprises a kind of protokaryon replication region, and for example ColE1 ori is used in the prokaryotic cell prokaryocyte and breeds, even this carrier is used to express in the cell type of other non-protokaryons.Carrier also can comprise a kind of suitable promotor, and for example a kind of gene that can instruct is by the prokaryotic promoter of the bacterial host cell of its conversion such as expression in escherichia coli (transcribe and translate).

Promotor is a kind of expression controlling elements, and it is by allowing RNA polymerase to form in conjunction with the dna sequence dna of also transcribing.Usually provide in plasmid vector with the promoter sequence of the host bacterium coupling of giving an example, this plasmid contains restriction site easily, is used for dna fragmentation of the present invention and inserts.

Typical prokaryotic vector plasmid is pUC18, pUC19, pBR322 and can be from BioradLaboratories (Richmond, CA, the USA) pBR329 of Huo Deing and can be from Pharmacia, Piscataway, NJ, the pTrc99A and the pK223-3 of USA acquisition.

A kind of typical mammalian cell vector plasmid is can be from Pharmacia, Piscataway, NJ, the pSVL that USA obtains.This carrier uses the SV40 late promoter to drive the expression of clone gene, and high expression level appears at and produces the antigenic cell of T, for example the COS-1 cell.

An example of induction type mammalian expression vector is pMSG, and it also can obtain from Pharmacia.This carrier uses the expression of the terminal repetition glucocorticoid inducible type promoters driven clone gene of mouse mammary tumour virus length.

Useful yeast plasmid carrier is pRS403-406 and pRS413-416, and they generally can be from Stratagene Cloning Systems, La Jolla, and CA 92037, and USA obtains.Plasmid pRS403, pRS404, pRS405 and pRS406 are yeast integrative plasmid (YIps), and are integrated with yeast selective marker HIS3, TRP1, LEU2 and URA3.Plasmid pRS413-416 is yeast centromeric plasmid (YCps).

Several different methods has been handled with carrier DNA by development and has been connected by complementary sticky end.For example, complementary homology polymer sequence can be added the dna fragmentation that will insert carrier.Make carrier and DNA by the hydrogen bonded between the complementary homology polymer tail then, form recombinant DNA molecules.

Synthetic, the joint that contains one or more restriction sites can be dna fragmentation the another kind of method that provides are provided with carrier.Handle with phage T4 archaeal dna polymerase or e. coli dna polymerase I with the dna fragmentation of restriction digest preparation by early stage introduction, these two kinds of enzymes can be removed 3 ' outstanding strand end by its 3 '-5 ' exonuclease activity, and pass through its polymerase activity with 3 ' recessed end-filling.

Therefore these active combination results the dna fragmentation of flat end.With flat terminal fragment and the excessive greatly linkers incubation in the presence of enzyme of mole number, wherein enzyme is catalysis to put down the enzyme that terminal dna molecular connects, for example phage T4 ligase enzyme then.Like this, the product of reaction is exactly the dna fragmentation that has the polymeric joint sequence endways.Cut these dna fragmentations with suitable restriction restriction endonuclease then, and be connected with the expression vector that cuts with the enzyme that can produce the end that is complementary with the end of dna fragmentation.

The synthetic linker that contains various restriction sites can be bought from multiple source, comprises International Biotechnologies Inc, New Haven, CN, USA.

A kind of Perfected process of modifying the DNA of coding polypeptide of the present invention is to use polymerase chain reaction according to (1988) " science " 239:487-491 disclosed methods such as Saiki.

In this method, the both wings of enzymatic amplification DNA are two species specific primers, and they self will be integrated into the DNA of amplification.Said Auele Specific Primer can contain restriction enzyme recognition site, is used to use method well known in the art to clone into expression vector.

Aspect the 4th, the present invention relates to polynucleotide carrier works transformed host cells of the present invention.Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell.Bacterial cell is preferred prokaryotic cell prokaryocyte, and is generally intestinal bacteria, for example can be from Bethesda ReserchLaboratories Inc., Bethesda, MD, the DH5 that USA obtains, and can be from Rockville, MD, the RR1 that the American type culture collection of USA (ATCC) obtains.Preferred eukaryotic host cell comprises yeast and mammalian cell, preferably from mouse, rat, monkey or human fibroblast cell line's vertebrate cells.Yeast host cell comprises YPH499, YPH500 and YPH501, and they generally can be from Stratagene CloningSystems, La Jolla, and CA 92037, and USA obtains.Preferred mammalian host cell comprises Chinese hamster ovary (CHO) the cell CCL61 that can obtain from ATCC, the NIH Swiss mouse protoblast NIH/3T3 cell CRL1658 that can obtain from ATCC and the monkey kidney deutero-COS-1 cell CRL1650 that can obtain from ATCC.

Aspect the 5th, the present invention relates to the purposes of polynucleotide carrier works of the present invention in the preparation medicinal composition.Specifically, the preparation vaccine is used for the treatment of or prophylaxis of tumours, fowl tumour preferably, especially those and carcinogenic avian viruses such as MDV, ALV, the rous-associated virus tumour relevant with the reticuloendothelial cell virus of proliferation.

In bird, with the solution of polynucleotide carrier works of the present invention zygote is carried out that injection is a kind of mode of administration easily in the ovum.

Transform proper host cell with dna structure thing of the present invention and finish by the method for knowing, and common type according to used carrier.Concerning the conversion of prokaryotic host cell, be seen in as (1989) " molecular cloning laboratory manual " Cold Spring Harbor Laboratory such as (1972) " PNAS " 69:2110 such as Cohen and Sambrook, ColdSpring Harbor, NY.The conversion of yeast cell is introduced among the NY at (1986) such as Sherman " yeast genetics method, laboratory manual " Cold Spring Harbor.The method of Beggs (1978) " nature " 275:104-109 is also useful.For vertebrate cells, can be to the transfection useful reagent of these cells such as calcium phosphate and DEAE-dextran or Liposomal formulation from Stratagene Cloning Systems or Life Technologies Inc., Gaithersburg, MD, 20877, USA obtains.

Electricity transforms and also can be used for transformant, and comes transformed yeast cell, bacterial cell and vertebrate cells to know in this area with it.

For example, various bacteria can transform with the method for introducing among (1988) " molecular microbiology " 2:637-646 such as Luehansky, and this article is hereby incorporated by.Using the every cm of 6250V that the DNA-cell mixture that is suspended in 2.5X PEB is carried out electricity at 25 uFD transforms, generally can obtain the transformant of maximum quantity.

The method of electricity transformed yeast is at Becker ﹠amp; Introduce among Grarente (1990) " Enzymology method " 194:182.

Transform successful cell, promptly contain the cell of dna structure thing of the present invention, the available technology of knowing is identified.For example, the cell that produces by importing a kind of expression structure thing of the present invention can be cultivated and produce polypeptide of the present invention.But collecting cell and cracking, and use the existence that detects target dna among its DNA such as the method among (1985) " biotechnology " 3:208 such as Southern (1975) " molecular biology magazine " 98:503 or Berent.In addition, can use antibody to press the existence that the introduction here detects target protein in the supernatant.

Except the existence of direct detection recombinant DNA, when recombinant DNA can instruct protein expression, successful conversion can prove by the immunological method of knowing.For example, can produce the albumen that shows suitable antigen with a kind of expression vector success cell transformed.The cell sample that collection may transform uses suitable antibody test target protein.

Therefore, except transformed host cell itself, the present invention also comprises the culture of these cells in a kind of nutritional medium, preferably a kind of monoclonal culture (clone's homogeneous), or by mono-clonal culture deutero-culture.

The non-limiting example that comprises all respects of the present invention will be introduced referring to figs. 1 to 7.

Fig. 1: the expression of plasmid PI.18, this plasmid contain the EcoRV site that an antigenic open reading frame of AV37 (ORF) can be inserted.

Fig. 2: fowlpox virus transferring plasmid (pEFL929), it contains the SmaI site that an AV37 ORF can insert.

Fig. 3: HVT transferring plasmid (pVECo4), it contains the EcoRV site that an AV37 ORF can insert.

Fig. 4: baculovirus transferring plasmid (pACYM1), it contains the SmaI site that an AV37 ORF can insert in multiple clone site (mcs)

Fig. 5 (a): the dna sequence dna of coding AV37 antigen and the antigenic aminoacid sequence of ripe AV37.

Fig. 5 (b): the antigenic aminoacid sequence of ripe AV37.

Fig. 6: the result who shows DELFIA.Salmonellas Ab represents that as positive control DELFIA is

normal.Reference number

1566,1568 and 1571 is the number of rings (2) with fowl of lymphadenomatous toxicity MDV infection; 391V and PK/2365 are for using the hyperimmune fowl of toxicity MDV.

Fig. 7: demonstration absorbs the DELFIA result behind the AV37 albumen in advance.Reference number 391V and 1599 expressions have the fowl that lymphadenomatous toxicity MDV infects.

Table 1-genetic code and the name of single-letter amino acid (are selected from Old ﹠amp; Primrose, " Principles of gene manipulation ", the third edition, 1985,7,346 pages of appendix, Blackwell ScientificPublications, Oxford, UK.)

5 '-OH does not hold base	Middle base				3 '-OH terminal bases
	Middle base					U	?C	?A	?G
	?U	?Phe ?Phe ?Leu ?Leu	?Ser ?Ser ?Ser ?Ser	?Tyr ?Tyr ?Stop ?Stop		U	?C	?A	?G	?Cys ?Cys ?Stop ?Trp	?U ?C ?A ?G
?C	?U	?Phe ?Phe ?Leu ?Leu	?Ser ?Ser ?Ser ?Ser	?Tyr ?Tyr ?Stop ?Stop	?Leu ?Leu ?Leu ?Leu	?Pro ?Pro ?Pro ?Pro	?His ?His ?Gln ?Gln	?Arg ?Arg ?Arg ?Arg	?U ?C ?A ?G	?Cys ?Cys ?Stop ?Trp	?U ?C ?A ?G
?C	?A	?Ile ?Ile ?Ile ?Met	?Thr ?Thr ?Thr ?Thr	?Asn ?Asn ?Lys ?Lys	?Leu ?Leu ?Leu ?Leu	?Pro ?Pro ?Pro ?Pro	?His ?His ?Gln ?Gln	?Arg ?Arg ?Arg ?Arg	?U ?C ?A ?G	?Ser ?Ser ?Arg ?Arg	?U ?C ?A ?G
?G	?A	?Ile ?Ile ?Ile ?Met	?Thr ?Thr ?Thr ?Thr	?Asn ?Asn ?Lys ?Lys	?Val ?Val ?Val ?Val ^a	?Ala ?Ala ?Ala ?Ala	?Asp ?Asp ?Glu ?Glu	?Gly ?Gly ?Gly ?Gly	?U ?C ?A ?G	?Ser ?Ser ?Arg ?Arg	?U ?C ?A ?G

^aIf fasten the preparation of hybridoma of preparation secretion mAb AV37 of monoclonal antibody AV37 of a kind of host antigen of expression for marek disease disease virus transformant at original position coding Met alanine A leucine L arginine R lysine K asparagine N methionine M aspartic acid D phenylalanine F cysteine C proline P cystine C serine S glycine G threonine T glutamic acid E tryptophan W glutamine Q tyrosine Y histidine H valine V isoleucine I

From MD tumour isolated cell, vitro culture, the clone [Powell of preparation immortalization, L.N., Payne, L.N., Frazier, J.A. and Rennie, M.C. (1975) " carcinogenic and simplexvirus II ", de-Th é, G.Epstein, M.A. and zurHausen, H. (editor) IARC Scientific Publications no.11, Lyon].From the MDV cell transformed is HP9[Payne, L.N. (1981) " international journal of cancer " 28:757-766] get 10 ⁷Cell is injected to an adult BALB/c mouse through the intraperitoneal approach with Quli A.After 4 weeks, be MDCC-HP89[Payne from the MDV cell transformed, L.N. (1981) " international journal of cancer " 28:757-766] get 10 ⁷Cell, this mouse is given in injection under the condition of identical approach at no adjuvant.8 all backs are with 10 ⁷The MDCC-HP89 cell repeats this step.After 6 weeks, by intravenous route injection 10 ⁷The MDCC-HP89 cell, and at 1 day after peritonaeum approach injection 10 ⁷The MDCC-HP89 cell is to strengthen immune Response of Mice.Put to death mouse after 3 days, take out spleen, the preparation splenocyte.Use polyoxyethylene glycol that splenocyte and NS1 myeloma cell are merged and prepare hybridoma [Kohler, G and Milstein, C. (1975) " nature " 256:495-497].The clone hybridization knurl, a hole--emiocytosis one strain antibody among-349 (AD4a), this antibody when being used for flow cytometry analysis, can with at least 90% MDCC-HP9 or-HP89 clone produces the male immune response, and identification is less than 10% thymocyte.Select this clone, and carry out the clone of next round, screening is to the positive reaction of MDV transformation cell lines cell.Selected hybridoma produces positive response to HP9 and HP89 cell line cell, but to not replying from the lymphocyte of the fowl that does not infect.The hybridoma of this two time cloning is named as AV37, and is preserved in ECACC on March 3rd, 1998 according to budapest treaty, and preserving number is 98030304.Use mAb AV37 to identify and express the antigenic cell of AV37

AV37 clone excretory monoclonal antibody (mAb AV37) can be discerned a kind of antigen that exists on all MD tumour deutero-cell line cells basically, except clone MDCC-MSB-1[Yamada M., Matsuda, H. and Hii, S. (1983) GANN, 74:502-508].AV37 antigen does not show expression (spleen, thymus gland, the fabricius bursa or marrow) on from the lymphocyte that does not infect fowl.As if but this monoclonal antibody can produce weak positive response (＜0.1% colony) to the minority peripheral blood lymphocyte.Stimulate lymphocyte and adding can increase with the phytohemagglutinin concanavalin A and express the antigenic cell proportion of AV37 (up 6%) from the lymphoblastic conditioned medium of activatory.After MDV infected, at metainfective the 3rd day and the 7th day, antigen detected on 2% peripheral blood lymphocyte.After the infection the 5th day on the splenocyte and on thymocyte, also detecting this antigen after the infection on the 12nd day.

AV37 antigen exists in the different cell parts (2-70%) of different MD tumours.These AV37 ⁺Cell is scattered in tumor tissues, but trends towards at different region clusterings.Flow cytometry analysis showed, most AV37 ⁺Tumour cell is CD4 ⁺, and express high-caliber MDV gene product meq, and it is a kind of alkaline leucine zipper protein with transcription factor activity, but does not have the viral protein that participates in lytic infection---pp38 and gB.A high proportion of CD4 ⁺AV37 ⁺Cell is shown in cell cycle S-G ₂The M phase, but have only small part to have little diploid DNA, this shows the active propagation of this cell colony, rather than experience activation inductive programmed cell death.Meq is external relevant with the blocking-up programmed cell death, and this illustrates the AV37 in tumour ⁺Cell is an immortalization, therefore is likely superfluous natural disposition transformant.A further important discovery is, the antigen of AV37 monoclonal antibody identification is fastened expression in other fowl oncovirus cell transformed, and this comprises retrovirus such as avian leukosis virus and reticuloendothelial cell virus of proliferation (seeing Table 2).This shows that AV37 antigen is a stud bird tumour specific antigen.

Table 2-mAbs AV37 is to the reactivity of avian cell lines MATSAs

Clone	Conversion reagent ¹	Fowl strain system ²	The tumour source	Reference or source ³	AV37
Clone	Conversion reagent ¹	Fowl strain system ²	The tumour source	Reference or source ³	AV37	?CHCC-OU2 ?LSCC-1104 ?LSCC-DT40 ?LSCC-DT95 ?LSCC-HP46 ?LSCC-RP9 ?MDCC-HP1 ?MDCC-HP3 ?MDCC-HP9 ?MDCC-HP10 ?MDCC-HP18 ?MDCC-HP28 ?MDCC-HP89 ?MDCC-MSB-1 ?MDCC-RP1 ?RECC-IAH16	?MNNG ?ALV ?RAV-1 ?RAV-1 ?ALV-HPRS-F42 ?RAV-2 ?MDV-HPRS-16 ?MDV-HPRS-16 ?MDV-HPRS-16 ?MDV-HPRS-16 ?MDV-HPRS-16 ?MDV-HPRS-16 ?MDV-HPRS-16 ?MDV1-BC1 ?MDV-JM ?REV-T	External RPRL 15I HL SC HL SC RPRL 15I RPRL 15Ix7 RIR RIR RPRL 7 RIR RPRL 6 RPRL 6 RIR RPRL 6	-bursa of farbricius bursa of farbricius bursa of farbricius spleen+liver transfer operation tumour ovary ovary spermary ovary ovary ovary ovary spleen transplantation tumour the bursa of farbricius	?Ogura?et?al.，1984 ?Hihara?et?al.，1974 ?Baba?et?al.，1985 ?Baba?et?al，1985 ?Nazerian，1986 ?Okazaki?et?al.，1980 ?Powell?et?al.，1974 ?Payne?et?al.，1981 ?Payne?et?al.，1981 ?Payne?et?al.，1981 ?Payne?et?al.，1981 ?Payne?et?al.，1981 ?Nazerian，1987 ?Akiyama?and?Kato，1974 ?Nazerian?et?al.，1977 ?Shaw，1997	????- ????+ ????- ????- ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????- ????+ ????+

The keyword 1 ALV=avian leukosis virus of table 2; The MDV=marek's disease virus; MNNG=

N-methyl-N'-nitro-N-nitroso-guani dine; The RAV=rous-associated virus; The REV=RE

Cell proliferation virus.2???HL?SC＝Hyline?Poultry?Farms，Dallas?Centre，IA，USA；RIR-Rhode

Island Red; Poultry research laboratory, RPRL-zone, East Lansing,

MI，USA.3???Oghura，H.et?al.(1984)GANN，75，410-414；

Hihara，H.et?al.(1974)Natl.Inst.Animal?Health?Quart.14，163-173；

Baba, T.W. (1985) " virusology " 144,139-151;

Nazerian, K.et al. (1977) avian disease 21,69-76;

Okazaki, W.et al. (1980) bird pathology 9,311-329;

Powell, P.C.et al. (1974) " nature " 251,79-80;

Payne, L.N.et al. (1981) " international journal of cancer " 28,757-766;

Nazerian, K. (1987) bird pathology 16,527-544;

Akiyama，Y.and?Kato，S.(1974)Biken，17，105-116；

Shaw, I. (1997) PhD thesis, the antigenic purifying of the antigenic evaluation of University of London.AV37 AV37

AV37 antigen is from the HP9 cell purification, and the surface protein of this cell is according to Vainio, O., and Riwar, B., Brown, M.H. and Lassila, " method that IMMUNOLOGY KEY WORDS INDEX 147:1593-1599 introduces is used newborn catalase Na to O. (1991) ¹²⁵I has carried out mark.Cell surface protein dissolves with the 2%NP40 lysis buffer that contains proteinase inhibitor.The radiolabeled antigen of forming a kind of soluble immune complex with AV37 antibody precipitates with the anti-rat immune globulin antibody mediated immunity of the rabbit that combines streptococcus protein G.With immunoprecipitation with the damping fluid that contains or do not contain mercaptoethanol dissociate [Laemmli, U.K. (1990) " nature " 227:680-685].When analyzing with sds polyacrylamide gel electrophoresis, under reduction and non-reduced condition, radiolabeled antigen all shows the relative molecular weight with 75kDa.The clone of AV37 gene and order-checking

(i) express the antigenic gene of being discerned by the AV37 monoclonal antibody and clone from a complementary DNA (cDNA) library, this library is from a kind of cell preparation of MDV transformation cell lines.The method of using Chirgwin etc. is from HP9 cell preparation messenger RNA(mRNA) [Chirgwin, J.M.Przbyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) " biological chemistry " 18:5294-5299], and with oligomerization (dT) chromatogram purification.Double-stranded DNA is connected with non-self complementary BstX1 joint, carries out the molecular weight size separation, and insert plasmid pCDM8[Seed, B. (1987) " nature " 329:840-842] with agarose gel electrophoresis.The DNA electricity that connects is transformed intestinal bacteria MC1061/p3[Dower, W.J., Miller, J.F. and Ragsdale, C.W. (1988) " nucleic acid exploration " 16:6127-6145].Plasmid prepares [Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) " molecular cloning laboratory manual ", Cold SpringHarbor, New York] by the blended transformed clone with standard method.The COS-7 cell transfecting carries out [Metzelaar in suspension or on adherent cell, M, Winjgaard, P.L.J., Peters, P., Sixma, J.J., Nieuwenhuis, H.K. and Clevers, H.C. (1991) " journal of biological chemistry " 266:3239-3245]. with the transfectional cell of monoclonal antibody AV37 screening acetone fixed and reclaim plasmid from the cell of positive staining and carry out [Horst, M., Wijngaard by the introduction of Horst, P.L.J., Metzelaar, M., Bast, E.J.E.G. and Clevers, H.C. (1991) " nucleic acid exploration " 19:4556].This AV37 ⁺The clone is named as pMAT1.A kind of monoclonal antibody AV7 that contrasts can not detect the staining cell of using the pMAT1 transfection, even these transfectional cells can dye consumingly with monoclonal antibody AV37.Use PRISM ^TMReady ReactionDye Deoxy ^TMTerminator cycle sequencing kit (Applied Biosystems) checks order to the pMAT1 clone.Measure the full sequence of every chain of this clone.Sequence data is analyzed [Devereux, J., Haeberli, P. and Smithies, O. (1984) " nucleic acid exploration " 12:387-395] with Wisconsin Package software (Genetics Computer Group).Complete nucleic acid and aminoacid sequence are shown in Fig. 5 (a).UW-GCG software package [Devereux, J., Haeberli, P. and Smithies, O. (1984) " nucleic acid exploration " 12:387-395] is used to search various DNA data.

Be unexpectedly, use the Southern engram analysis not detect the antigenic gene of coding AV37 on MDV DNA, this shows that it is not a kind of virogene.But use chicken lymphocyte that RT-polymerase chain reaction (RT-PCR) never infects and hatch this gene that increased in total RNA extract of egg cell of 5 days.This shows that AV37 is a carcinomebryonic antigen, the carcinomebryonic antigen of introducing in the similar Mammals [summary: Boon, T. and Old, L.J. " Immunization Update comment " (1997) 9:681-683; Van den Eyde, B. and vand der Bruggen, P. " Immunization Update comment " (1997) 9:684-693].

The AV37 monoclonal antibody can also be discerned the lymph matricyte system that transforms preparation with other oncoviruses.See Table the introduction of 2 (above) with the positive response of clone acquisition.This shows that AV37 antigen is a kind of mark that transforms, and is present in the tumour cell that is produced by other oncoviruses.

(ii) express the antigenic gene of being discerned by the AV37 monoclonal antibody and from a complementary DNA (cDNA) library, clone, the lymph matricyte system MDCC-HP9 preparation that this library transforms from the marek disease disease virus.All MDCC-HP9 cells are all expressed very high-caliber AV37 antigen.The method of using Chirgwin etc. is from HP9 cell preparation messenger RNA(mRNA) [Chirgwin, J.M.Przbyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) " biological chemistry " 18:5294-5299], and with oligomerization (dT) chromatogram purification.Double-stranded DNA is connected with non-self complementary BstX1 joint, carries out the molecular weight size separation with agarose gel electrophoresis, and insert plasmid vector pCI-nx, this plasmid is by IAH, and doctor J.Young of Compton gives.PCI-nx is commercialization plasmid pCI-neo (Promega, Madison, varient Wisconsin).This library electricity is transformed intestinal bacteria MC1061/p3[Dower, W.J., Miller, J.F. and Ragsdale, C.W. (1988) " nucleic acid exploration " 16:6127-6145].Plasmid prepares [Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) " molecular cloning laboratory manual ", Cold Spring Harbor, New York] by the blended transformed clone with standard method.The COS-7 cell transfecting carries out [Metzelaar, M, Winjgaard, P.L.J., Peters, P., Sixma, J.J., Nieuwenhuis, H.K. and Clevers, H.C. (1991) " journal of biological chemistry " 266:3239-3245] in suspension.With the bag by the Dynabeads  M280 of sheep anti mouse immunoglobulin (Ig) (Dynal, Oslo, Norway) with AV37 antibody incubation, the washing and with transfection the COS-7 cell incubation in HP9 library.Then they are carried out magnetic separation [O ' Regan, M.N., Parsons, K.R., Tregaskes, C.A. and Young, J.R. (1999) " immunogenetics " 49:68-71] by the introduction of O ' Regan etc.Carry out 3 and take turns such selection and enrichment.24 positive colonies have been separated.Check order to can when COS-7 cell or chick embryo fibroblast are advanced in transfection, expressing the antigenic clone of AV37.(Milton Keynes UK) uses LICOR 4200 systems to check order to the AV37 gene by MWG-Bicotech.The clone is carried out the complete order-checking of two chains.

UW-GCG software package [Devereux, J., Haeberli, P. and Smithies, O. (1984) " nucleic acid exploration " 12:387-395] is used to search various DNA data.These searches show that AV37 is a member of tumor necrosis factor receptor super family.

Sophisticated AV37 antigen is the 1 type membranin of a kind of 446aa, and is the most similar with mouse CD30 to (1) people, and the whole amino acid whose identity of it and people and mouse CD30 is approximately 32%.The antigenic aminoacid sequence of AV37

For directly measuring aminoacid sequence, directly separate AV37 antigen from MDCC HP9 clone.After the membranin lysate to the HP9 cell carries out immunoblotting (Western trace), the reaction of the band of an AV37 antibody and a 70kDa.The AV37 mono-clonal is prepared by cultivate hybridoma in containing the substratum that has or not immunoglobulin (Ig) serum.AV37 immunoglobulin (Ig) albumin A (Pharmacia) purifying, then use standard techniques coupling [Coligan, J.E., Kruisbeek with CNBr-activatory Sepharose (Pharmacia), A.M., Marulies, D.H., Shevach, E.M. and Strober, the 2nd edition the 8th chapter of W (1999) " Immunization Update method ", suppl.29, John Wiley and Sons Inc.].The membranin lysate is from 10 ¹⁰The HP cell preparation, by the AV37-Sepharose post, wash-out contains the antigenic level part of AV37 with lysate.The level duplicate samples is carried out immunoblotting to be identified and contains the antigenic level of AV37 part.(after the membranin lysate to the HP9 cell carries out immunoblotting (Western trace), the reaction of the band of an AV37 antibody and a 70kDa.)。With part mixing of positive level, use acetone precipitation, and preparation is used to use Edman degraded carrying out N-end sequencing [seeing Niall, HD (1973) " Enzymology method " 2 volumes, 942-1010 page or leaf].Measure 7-terminal amino acids, this has proved the proteic prediction initiation site of ripe AV37 (amino acid 22).The purposes of the coding antigenic nucleic acid of AV37 in vector construction and immunization method and composition

1.AV37 the source of ORF

The coding antigenic open reading frame of AV37 (ORF) can use a kind of cDNA clone of pcr amplification (pMAT1.AV37) to obtain, and uses following primer.

Be applicable to that the primer that obtains the AV37 gene ORF is to giving an example:

Forward

5’-TGG?AAA?GGA?ACT?GGA?GTG-3’????????(SEQ?ID?No.6)

Oppositely

5’-GCA?AGC?TGT?GAA?TTA?GCC-3’????????(SEQ?ID?No.7)

Forward

5’-CTG?CTG?CTT?CTC?CAG?GAC?ATT?C-3’??(SEQ?ID?No.8)

Oppositely

5’-ATT?CCT?TTC?CCT?CCT?CTT?CCA?C-3’???(SEQ?ID?No.9)

Forward

5’-AGA?CTT?CAG?GAG?GAG?CAA?C-3’???????(SEQ?ID?No.10)

Oppositely

5’-GCT?TCA?CAC?ACC?TTC?TCA?G-3’???????(SEQ?ID?No.11)

These primers both wings that are designed to increase contain the ORF of the AV37 in EcoRV site.

Should be appreciated that every kind of primer also can be used alone as hybridization probe (as the radio-labeled probe), detect the varient of the AV37 nucleotide sequence of Fig. 5 (a) (SEQ ID No.1) according to standard technique.2. the structure of expression plasmid

The PCR product that obtains above can digest with EcoRV, and fragment cloning is entered the EcoRV site of expression plasmid PI.18 (Fig. 1).The PI.18 plasmid is available from doctor J.S.Robertson, National Institute of Biological Standards and Control, SouthMimms, EN6 3QG, UK.It is derived by pUC plasmid [Sambrook etc. (1989) " molecular cloning laboratory manual " Cold Spring Harbor Laboratory, Cold SpringHarbor, NY], and is inactivity.It has been used to by genetic engineering expression alien gene in Mammals and avian cell.Expression is driven by the instant early promoter of HCMV, and optimizes by contain an intron between promotor and cloning site.Transcribe signal terminating by CMV polyA.The analysis of the segmental direction available constraints of AV37 enzyme detects, and with the order-checking direction of justifying.The recombinant plasmid (PI.18-AV37) that obtains but chick embryo fibroblast is advanced in transfection, and the expression of AV37 antigen under the control of CMV I.E. promotor can be used mAb AV37 to carry out immunofluorescence method to detect.This plasmid can be directly used in to be inoculated chicken immune.It is preferably at first at Qiagen ^TMPurifying on the anionite-exchange resin.Methods of vaccination

Chicken can carry out immunization in a plurality of sites of leg and chest with the recombinant plasmid dna (30-100ug) of various dosage.Booster immunization can (for example 15 days) carry out after appropriate intervals, and with the toxicity strain (RB1B) of a kind of MDV chicken is attacked.Only with the chicken of plasmid PI.18 immunity with comparing.3. the structure of recombinant of fowlpox virus

In the present embodiment, come a kind of known epiornitic seedling is modified by the nucleotide sequence that inserts a kind of AV37 of coding of the present invention.The reagent that is produced thereby contain the composition of a kind of nucleotide sequence/polypeptide of the present invention and a kind of known epiornitic seedling.

The EcoRV fragment that contains AV37 ORF can obtain by EcoRV digestion from PI.18-AV37, and the clone enters the SmaI site of fowlpox virus transferring plasmid pEFL29 (Fig. 2).The correct direction of AV37 gene can detect according to top introduction.Chick embryo fibroblast is advanced in this works transfection, this cell according to people's (1994) [" virological method magazine " 50:185-196] such as people such as Yu (1994) [" vaccine " 12:227-237] and Li introduction transfection in advance the FP9 strain of fowlpox virus.The recombinant virus filial generation can identify that Bluogal is a kind of substrate of beta-galactosidase enzymes by form blue look plaque when having Bluogal (Sigma).But the recombinant virus of plaque purifying some amount, and use monoclonal antibody AV37 to detect the expression of AV37 by immunofluorescence technique.Methods of vaccination

Chicken can pass through i/m or intradermal vaccination, uses cut on the wing usually, and can use different vaccination dosage (for example 10 ⁶-10 ⁷Plaque forming unit) recombinant virus.They can use identical dosage and identical approach to carry out the 2nd immunity after 14 days to carry out booster immunization.The inoculation chicken and with the immunity of FP9 carrier to available RB1B virus attack after impinging upon 6 days.4. the structure of herpes turkey virus (HVT) recombinant chou

Can clone the into EcoRV site of HVT transferring plasmid pVECo4 (Fig. 3) [Sondermeijer etc. (1993) " vaccine " 11:349-358] by the EcoRV fragment that contains AV37 ORF that top introduction obtains.The correct direction of AV37 gene can detect according to top introduction.This works and infectious HVT DNA are advanced CEF (Morgan etc. (1992) " poultry disease " 36:858-870 by introducing cotransfection; Ross etc. (1993) " general virology magazine ", 74:371-377).But picking some amount virus plaque, and according to top introduction, the infection CEF that expresses AV37 by screening identifies recombinant virus.Methods of vaccination

Chicken can inoculate the recombinant hvt or the parent HVT of 1000 to 5000 plaque forming units by i/m, and can use the RB1B virus attack after 6 days.5. baculovirus recombinant chou

The EcoRV fragment that contains AV37 ORF can be cloned the SmaI site [Matsuura, Y. etc. (1987) " general virology magazine " 68:1233-1250] in the multiple clone site (mcs) of baculovirus transferring plasmid PACYMI (Fig. 4) into.The baculovirus DNA of recombinant plasmid and linearization can be according to Matsuura, and the cotransfection of introducing of Y. etc. (1987) advances the SF9 insect cell.Plaque purifying progeny virus, and detect them by the immunofluorescence method of introducing above and in the SF9 cell, express the antigenic ability of AV37.Methods of vaccination

Infect the extract of SF9 cell or can be used for immunization with the AV37 antigen of affinity chromatography purifying.For primary vaccination, antigen can emulsification in the Fu Shi Freund's complete adjuvant.After suitable interval (as 3 weeks), use Freund further to inoculate, adjuvant is not used in last inoculation.After inoculating for 1 week the last time, with the chicken of RB1B virus attack inoculation.With the chicken of the insect cell extract immunity of having infected wild-type baculovirus in contrast.

In all vaccination strategies, the clinical symptom of the MD that the degree of protection that provides can be by observing acceptor fowl and tumour form to be measured.Can collect blood sample after the time in suitable interval, screen the existence of the antigenic antibody of anti-AV37.All acceptor fowl can be put to death after week at for example 15-26, and performs an autopsy on sb, and detects the sign of tumour.6. the further method of immunization

Chicken can inoculate by approach in the ovum.On 18 days the shell of zygote of hatching, hole, will be up to allantoic cavity or the amniotic cavity that reorganization AV37 antigen in HVT, fowlpox virus carrier or the baculovirus vector or plasmid DNA form are injected into the chicken embryo of growing that contain of 0.1ml.Very useful automatization ovum injection device can be from Embrex Inc., and USA obtains.

The further inoculation of no adjuvant can be after suitable interval (for example 2-3 week) chicken of hatching through suitable approach.The chicken of inoculation inoculates the back RB1B virus attack of 1 week the last time.The chicken of the extract immunity of the insect cell that infects with HVT, fowlpox virus or with wild-type baculovirus is as separately contrast.7. in the chicken that MDV infects, induced a kind of obvious immunne response at AV37

Obtain serum from following approach: (1) has been used a kind of genetic resistance chicken of toxicity MDV superingection to infect with toxicity MDV with (2) and has been produced lymphadenomatous susceptible chicken.These serum in immunosorbent adsorption test with the AV37 antigen positive of purifying reaction (Fig. 6).On the contrary, from the control serum of anosis chicken in immunosorbent adsorption test not with the AV37 antigen-reactive of purifying.The chicken that this explanation MDV infects and the chicken of immunity have produced the antigenic antibody at AV37.In addition, when the preincubation of the AV37 of these serum and purifying antigen, antibody is got rid of from serum, positive reading disappearance (Fig. 7).The experiment summary:

The chicken of strain 6 has genetic resistance to MD, and they carry out hyperimmunization by the carcinogenic strain of a kind of MDV of duplicate injection (HPRS-16 strain).Collect serum, and be stored in-20 ℃ in order to analyzing.In addition, the red chicken in the Rhode Island of MD susceptible is infected with MDV (HPRS-16 strain), and after producing horsepower creutzfeldt jakob disease lymphoma, collect serum.Control serum is collected from the chicken of the identical strain that is in anosis environment.

The AV37 mono-clonal is by cultivating the hybridoma preparation in containing the substratum that has or not immunoglobulin (Ig) serum.Immunoglobulin (Ig) albumin A (Pharmacia) purifying, then use standard techniques coupling [Coligan, J.E., Kruisbeek with CNBr-activatory Sepharose (Pharmacia), A.M., Marulies, D.H., Shevach, E.M. and Strober, the 2nd edition the 8th chapter of W (1999) " Immunization Update method ", suppl.29, John Wiley and Sons Inc.].The membranin lysate is from 10 ¹⁰The HP cell preparation, by the AV37-Sepharose post, wash-out contains the antigenic level part of AV37 with lysate.The level duplicate samples is carried out immunoblotting to be identified and contains the antigenic level of AV37 part.

Spend the night by the antigenic lysate of the AV37 that contains purifying at 4 ℃ of incubations, AV37 is antigen coated in the hole of plastics titer plate.Wash plate is also sealed with containing the sealing damping fluid of bovine serum albumin as closed reagent.Dilution from the hyper-immuneserum of MDV immunity chicken, carry the MDV infected chicken serum of tumour or from the control serum of infected chicken not, and in the hole of AV37 bag quilt incubation.With plate washing 3 times, then with the anti-chicken immune sphaeroprotein incubation that has engaged Europium, and be used to dissociate enhanced lanthanum fluorescent test (DELFIA) [Wood, P.﹠amp; Barnard, G. (1997) " principle of immunity test and put into practice " PriceDavid C.P. edits 17 chapters, Newman Macmillan, London].Compare hyper-immuneserum and sero-reaction positive (lanthanum fluorescence the is stronger) (see figure 6) that carries the MDV infected chicken of tumour with the serum of the chicken that infects from the beginning.

Whether for detecting that this replys is specific, and identical serum is being wrapped by preincubation in the hole of the AV37 antigen of purifying or horse serum.Then sample is used for DELFIA.Hyper-immuneserum can not react (Fig. 7) with the serum that carries the MDV infected chicken of tumour in DELFIA, this shows that the AV37 specific antibody in the serum is removed.

＜110〉Institute of Animal Health＜120〉＜130〉INST-P20757PC＜140〉＜141〉＜150〉GB 9809070.7＜151〉1998-04-29＜160〉11＜170〉PatentIn Ver.2.0＜210〉1＜211〉1956＜212〉DNA＜213〉Gallus sp.＜220〉＜221〉＜222〉 ( 205 ) .. ( 1605 )＜400〉1tcgagaattc acgcgtggta cctctagaga tccctcgacc tcgagatcca ttgtgctgga 60aaggaactgg agtggccctg gacttggtga gcggacactg agcgaggctg gactggacgc 120tgggagggtc ccagacttca ggaggagcaa cctggctgtc agacctgcaa tgctgggaga 180tcaggaaagc taacgccact gcagatggct tcctgcagcc tgagactggg actgtggctc 240ctgctgcttc tccaggacat tcagggagcc ccacagccac cgttcacctc atctcattcc 300tgtgacacac tcaagaattg gttctatgat gaaacattag ggaggtgctg ttaccagtgc 360ccttcaggct atgctaaaaa gaaatcatgt cccatggatc cagatgaaga ctgcatgaga 420tgtggacctg agcaatacct gaatcagtcc ccaaagccac gatgtgatgc ttgtgtgtta 480tgcaccaaag aatttgacct tgtggagaag gccccctgct ccttcaattc cagccgggtg 540tgtgagtgtc gaccagggat gttttgccag actgctgcta agaacacctg catgcgatgc 600cagcggcaca ctgcttgcaa gcctggtttt ggggtcaaaa tcagaggcac ttcagagact 660gatgtttcat gtgaggaatg ccctcctggg accttctctg accaaagctc cagcactgac 720gtctgcaagc cccacacgga ctgtgccaag ttgaacaaag tagcacaagg caaaggaaat 780gccacccatg atcaggtttg cacggaccaa ttgccctcct acctcacccc agacacctcc 840tccatcagaa tcaccaatga gacagatgac tctgacgtac tgaagcgtaa tgcaaacccg 900gtgacccttg ctagcatcct ttcaagtgcc acaaccgaaa ttcccggttc aactcccgag 960gaggaagctc tggctggcac ttctcccacc ttagccaagg gggaaacaac aacgagaggt 1020cttgttttct gggcagtggt tctctctgtg atggtgctac ctgtgggcat gctgtcattt 1080tggcaatgga aggtctgcaa gaaacggatc ttcatcctca aacaaaagcg ttctgatcta 1140gtggacaaat atgcaaagat cacactgacc actgacaaat gtccagaaga ggaggagtta 1200actgacagga gcctcccttt ggaaaccaac aacaacaact tgatctccag tgctgaaaaa 1260gcaggtagtc ctgttctgag tctgactgaa gtgacgcaga gcaatggaaa agcgccagat 1320ggccccattg attctcaagt gagagaccac acaaataatc agattggaaa aatattcatc 1380atgaacgccg ataccgttat tgtggggtct tcaaaaacgc ctggtatcaa gagctgcact 1440gctaggggat atgaaactga tgttgatctc caggaaaaga tggaagagga gctgtcaatg 1500cactatccag agcaggagac agaggttttt ccagggaatg atgtcatggt tcctgtggaa 1560gaggagggaa aggaattcca tcaccccacc acggccactg agaagtgatc gcctgctgag 1620aaggtgtgtg aagctacagc aacatccagt gacactaagc ctgaacccac actgagggag 1680gtaaacccag agtgtcttac acgacctgaa aactcacgta aagcaccaaa aacattcagc 1740ttatttcatc cagctaattg agaggatcat ccagaccact ggttcacatc aaacactttt 1800ccttgccctc ccagaatcat gctggaaaca aactggaatc aaagttacag aattcagagg 1860acttctggag gctaattcac agcttgcttt gtctgcatga agggatggaa ttaaaatggt 1920tactcctatt agctctgaaa aaaaaaaaaa aaaaaa 1956＜210〉2＜211〉467＜212〉PRT＜213〉Gallus sp.＜400〉2Met Ala Ser Cys Ser Leu Arg Leu Gly Leu Trp Leu Leu Leu Leu Leu 1 5 10 15Gln Asp Ile Gln Gly Ala Pro Gln Pro Pro Phe Thr Ser Ser His Ser

20??????????????????25??????????????????30Cys?Asp?Thr?Leu?Lys?Asn?Trp?Phe?Tyr?Asp?Glu?Thr?Leu?Gly?Arg?Cys

35??????????????????40??????????????????45Cys?Tyr?Gln?Cys?Pro?Ser?Gly?Tyr?Ala?Lys?Lys?Lys?Ser?Cys?Pro?Met

50??????????????????55??????????????????60Asp?Pro?Asp?Glu?Asp?Cys?Met?Arg?Cys?Gly?Pro?Glu?Gln?Tyr?Leu?Asn?65??????????????????70??????????????????75??????????????????80Gln?Ser?Pro?Lys?Pro?Arg?Cys?Asp?Ala?Cys?Val?Leu?Cys?Thr?Lys?Glu

85??????????????????90??????????????????95Phe?Asp?Leu?Val?Glu?Lys?Ala?Pro?Cys?Ser?Phe?Asn?Ser?Ser?Arg?Val

100?????????????????105?????????????????110Cys?Glu?Cys?Arg?Pro?Gly?Met?Phe?Cys?Gln?Thr?Ala?Ala?Lys?Asn?Thr

115?????????????????120?????????????????125Cys?Met?Arg?Cys?Gln?Arg?His?Thr?Ala?Cys?Lys?Pro?Gly?Phe?Gly?Val

130?????????????????135?????????????????140Lys?Ile?Arg?Gly?Thr?Ser?Glu?Thr?Asp?Val?Ser?Cys?Glu?Glu?Cys?Pro145?????????????????150?????????????????155?????????????????160Pro?Gly?Thr?Phe?Ser?Asp?Gln?Ser?Ser?Ser?Thr?Asp?Val?Cys?Lys?Pro

165?????????????????170?????????????????175His?Thr?Asp?Cys?Ala?Lys?Leu?Asn?Lys?Val?Ala?Gln?Gly?Lys?Gly?Asn

180?????????????????185?????????????????190Ala?Thr?His?Asp?Gln?Val?Cys?Thr?Asp?Gln?Leu?Pro?Ser?Tyr?Leu?Thr

195?????????????????200?????????????????205Pro?Asp?Thr?Ser?Ser?Ile?Arg?Ile?Thr?Asn?Glu?Thr?Asp?Asp?Ser?Asp

210?????????????????215?????????????????220Val?Leu?Lys?Arg?Asn?Ala?Asn?Pro?Val?Thr?Leu?Ala?Ser?Ile?Leu?Ser225?????????????????230?????????????????235?????????????????240Ser?Ala?Thr?Thr?Glu?Ile?Pro?Gly?Ser?Thr?Pro?Glu?Glu?Glu?Ala?Leu

245?????????????????250?????????????????255Ala?Gly?Thr?Ser?Pro?Thr?Leu?Ala?Lys?Gly?Glu?Thr?Thr?Thr?Arg?Gly

260?????????????????265?????????????????270Leu?Val?Phe?Trp?Ala?Val?Val?Leu?Ser?Val?Met?Val?Leu?Pro?Val?Gly

275?????????????????280?????????????????285Met?Leu?Ser?Phe?Trp?Gln?Trp?Lys?Val?Cys?Lys?Lys?Arg?Ile?Phe?Ile

290?????????????????295?????????????????300Leu?Lys?Gln?Lys?Arg?Ser?Asp?Leu?Val?Asp?Lys?Tyr?Ala?Lys?Ile?Thr305?????????????????310?????????????????315?????????????????320Leu?Thr?Thr?Asp?Lys?Cys?Pro?Glu?Glu?Glu?Glu?Leu?Thr?Asp?Arg?Ser

325?????????????????330?????????????????335Leu?Pro?Leu?Glu?Thr?Asn?Asn?Asn?Asn?Leu?Ile?Ser?Ser?Ala?Glu?Lys

340?????????????????345?????????????????350Ala?Gly?Ser?Pro?Val?Leu?Ser?Leu?Thr?Glu?Val?Thr?Gln?Ser?Asn?Gly

355?????????????????360?????????????????365Lys?Ala?Pro?Asp?Gly?Pro?Ile?Asp?Ser?Gln?Val?Arg?Asp?His?Thr?Asn

370?????????????????375?????????????????380Asn?Gln?Ile?Gly?Lys?Ile?Phe?Ile?Met?Asn?Ala?Asp?Thr?Val?Ile?Val385?????????????????390?????????????????395?????????????????400Gly?Ser?Ser?Lys?Thr?Pro?Gly?Ile?Lys?Ser?Cys?Thr?Ala?Arg?Gly?Tyr

405?????????????????410?????????????????415Glu?Thr?Asp?Val?Asp?Leu?Gln?Glu?Lys?Met?Glu?Glu?Glu?Leu?Ser?Met

420?????????????????425?????????????????430His?Tyr?Pro?Glu?Gln?Glu?Thr?Glu?Val?Phe?Pro?Gly?Asn?Asp?Val?Met

435?????????????????440?????????????????445Val?Pro?Val?Glu?Glu?Glu?Gly?Lys?Glu?Phe?His?His?Pro?Thr?Thr?Ala

450?????????????????455?????????????????460Thr?Glu?Lys465<210>3<211>16<212>PRT<213>Gallus?sp.<400>3Cys?Asp?Thr?Leu?Lys?Asn?Trp?Phe?Tyr?Asp?Glu?Thr?Leu?Gly?Arg?Cys??1???????????????5??????????????????10??????????????????15<210>4<211>22<212>PRT<213>Gallus?sp.<400>4Asp?Val?Met?Val?Pro?Val?Glu?Glu?Glu?Gly?Lys?Glu?Phe?His?His?Pro??1???????????????5??????????????????10??????????????????15Thr?Thr?Ala?Thr?Glu?Lys

20<210>5<211>22<212>PRT<213>Gallus?sp.<400>5Gln?Pro?Pro?Phe?Thr?Ser?Ser?His?Ser?Cys?Asp?Thr?Leu?Lys?Asn?Trp??1???????????????5??????????????????10??????????????????15Phe?Tyr?Asp?Glu?Thr?Leu

20<210>6<211>18<212>DNA<213>Gallus?sp.<400>6tggaaaggaa?ctggagtg??????????????????????????????????????????????18<210>7<211>18<212>DNA<213>Gallus?sp.<400>7gcaagctgtg?aattagcc??????????????????????????????????????????????18<210>8<211>22<212>DNA<213>Gallus?sp.<400>8ctgctgcttc?tccaggacat?tc?????????????????????????????????????????22<210>9<211>22<212>DNA<213>Gallus?sp.<400>9attcctttcc?ctcctcttcc?ac?????????????????????????????????????????22<210>10<211>19<212>DNA<213>Gallus?sp.<400>10agacttcagg?aggagcaac?????????????????????????????????????????????19<210>11<211>19<212>DNA<213>Gallus?sp.<400>11gcttcacaca?ccttctcag????????????????????????????????????????????19

Claims

1. an energy is by the purified polypeptide of hybridoma AV37 excretory monoclonal antibody identification, and wherein this hybridoma is deposited in the European zooblast preservation center (ECACC) that is positioned at Britain according to budapest treaty on March 3rd, 1998, and preserving number is 98030304.

2. the polypeptide of claim 1, wherein expression of polypeptides is being fastened by a kind of carcinogenic avian viruses cell transformed.

3. the polypeptide of claim 2, wherein the carinogenicity avian viruses is selected from marek disease disease virus, avian leukosis virus, rous-associated virus or reticuloendothelial cell virus of proliferation.

4. the polypeptide of claim 3, wherein virus is the marek disease disease virus.

5. a peptide species, each polypeptide of claim 1-4 preferably, wherein this polypeptide contains aminoacid sequence whole or fragment or the varient of Fig. 5 (b) (SEQ ID No.2).

6. the polypeptide of claim 5, it has and is selected from following AV37 amino acid sequence of polypeptide:

(a) NH ₂-QPPFTSSHSCDTLKNWFYDETL-COOH (SEQ ID No.5); With

(b) NH ₂-DVMVPVEEEGKEFHHPTTATEK-COOH (SEQ ID No.4); And/or

(c)NH ₂-CDTLKNWFYDETLGRC-COOH(SEQ?ID?No.3)。

7. at each the monoclonal antibody of polypeptide preparation of claim 1-6.

8. can be by the purified polypeptide of the monoclonal antibody of claim 7 identification.

9. separating nucleotide sequence or its varient of each or 8 polypeptide of coding claim 1 to 6.

10. DNA isolation sequence or its varient of each or 8 polypeptide of coding claim 1 to 6.

11. the dna sequence dna of claim 10, it comprises all or part of of the nucleotide sequence that is shown in Fig. 5 (a) (SEQ ID No.1).

12. contain each the polynucleotide carrier works of nucleotide sequence of claim 9 to 11.

13. contain the expression vector of the dna sequence dna of claim 10 or 11, wherein dna sequence dna is arranged in the carrier, and this dna sequence dna is expressed in a kind of proper host cell.

14. the expression vector of claim 13, wherein carrier comprises plasmid.

15. the expression vector of claim 13, wherein carrier comprises viral DNA.

16. the expression vector of claim 15, wherein viral DNA is from fowlpox virus, herpes turkey virus and/or baculovirus.

17. each carrier or claim 1 to 6 each or 8 the polypeptide purposes in a kind of medicinal composition of preparation of claim 12 to 16.

18. claim 12 to 16 each carrier or claim 1 to 6 each or 8 polypeptide at a kind of vaccine of preparation with the purposes in the composition.

19. the purposes of claim 17 or 18, the composition that is used to prepare treatment or prevents the fowl tumour.

20. the purposes of claim 18, wherein vaccine is at one or more carcinogenic avian viruseses.

21. the purposes of claim 20, wherein the carinogenicity avian viruses is selected from one or more in marek disease disease virus, avian leukosis virus, rous-associated virus or the reticuloendothelial cell virus of proliferation.

22. each purposes of claim 17 to 21 is used in combination with another kind of vaccine.

23. the purposes of claim 22, wherein another kind of vaccine is selected from HVT and fowlpox virus.

24. contain claim 1 to 6 each or 8 polypeptide and a kind of composition of pharmaceutically acceptable vehicle.

25. contain claim 1 to 6 each or 8 polypeptide and a kind of composition of adjuvant.

26. a method for preparing polypeptide is included in a kind of suitable cell and expresses each nucleotide sequence or its varient of claim 8 to 10, and purified polypeptide.

27. be deposited in ECACC according to budapest treaty on March 3rd, 1998, the hybridoma AV37 of UK, its preserving number are 98030304.

28. the hybridoma excretory monoclonal antibody of claim 27.

29. the monoclonal antibody of claim 7 or 28 is in a kind of purposes of identifying in polypeptide or its segmental method.

30. with each polynucleotide carrier works transformed host cells of claim 12 to 16.

31. be selected from the nucleotide sequence of following sequence:

5’-TGG?AAA?GGA?ACT?GGA?GTG-3’????????(SEQ?ID?No.6)

5’-GCA?AGC?TGT?GAA?TTA?GCC-3’????????(SEQ?ID?No.7)

5’-CTG?CTG?CTT?CTC?CAG?GAC?ATT?C-3’??(SEQ?ID?No.8)

5’-ATT?CCT?TTC?CCT?CCT?CTT?CCA?C-3’???(SEQ?ID?No.9)

5’-AGA?CTT?CAG?GAG?GAG?CAA?C-3’???????(SEQ?ID?No.10)

5’-GCT?TCA?CAC?ACC?TTC?TCA?G-3’???????(SEQ?ID?No.11)

32. one or more nucleotide sequences of claim 31 are as the purposes of primer in nucleic acid amplification reaction.

33. one or more nucleotide sequences of claim 31 are as the purposes of hybridization probe in the varient of the nucleotide sequence of test right requirement 9 to 11.

34. a method for preparing antibody comprises providing claim 1 to 6 each or 8 polypeptide or its fragment or varient; This polypeptide or its fragment or varient are delivered medicine to a kind of Mammals produce antibody response, and obtain antibody from this Mammals.

35. one kind prepares the method that can produce a kind of monoclonal hybridoma, comprising provides claim 1 to 6 each or 8 polypeptide or its fragment or varient; This polypeptide or its fragment or varient are delivered medicine to a kind of Mammals produce antibody response, from this Mammals, obtain a kind of antibody produced cell, and it and a kind of immortality cell are merged, form a kind of hybridoma of energy secrete monoclonal antibody.

36. pass through the antibody that the method for claim 34 obtains.

37. monoclonal antibody with the preparation of the hybridoma of claim 35.