CN106866796A

CN106866796A - The recombinant protein of the type S9 gene codes of GCRV II and its application

Info

Publication number: CN106866796A
Application number: CN201710067886.5A
Authority: CN
Inventors: 曾伟伟; 王庆; 王英英; 李莹莹; 石存斌; 任燕
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2017-02-07
Filing date: 2017-02-07
Publication date: 2017-06-20

Abstract

Recombinant protein and its application the invention discloses a kind of type S9 gene codes of GCRV II.The amino acid sequence of the recombinant protein such as SEQ ID NO:Shown in 2, the nucleotide sequence such as SEQ ID NO of the albumen are encoded:Shown in 1.The recombinant protein that the present invention is obtained has preferable immunogenicity, and compared with GCRV other structures albumen, the specific antibody titres that the immune animal of its induction produces are higher.Further experiment shows, with VP6 protein immunization grass carps, can induce grass carp and produce specific antibody higher, and can play certain immanoprotection action to the attack of GCRV velogen strain.Therefore, to the research and application of the type VP6 albumen of GCRV II, split hair hemorrhagic disease of grass carp new generation vaccine and immunological detecting kit are extremely important, for hemorrhagic disease of grass carp provides new effective solution route.

Description

The recombinant protein of the type S9 gene codes of GCRV II and its application

Technical field

The invention belongs to biological gene engineering field, and in particular to by the weight of the type S9 gene codes of GCRV II Group VP6 antigen proteins and its application.

Background technology

The hemorrhagic disease of grass carp caused by GCRV (Grass Crap Reovirus, GCRV) gives China grass carp Aquaculture causes huge economic losses, seriously governs the sound development of China's grass carp cultivation.GCRV has double capsid, virion Sub- average diameter be 60nm~70nm, icosahedral symmetry, without cyst membrane, genome is made up of 11 segmented double-stranded RNAs, root According to the size of its gene segment, its genome is divided into 3 groups by molecular size range, is respectively designated as L1-L3, M4-M6 and S7- S11.This 11 RNA sections can encode 12 kinds of polypeptide proteins, but specific which gene code structural proteins, which non-knot of coding If the function of structure albumen and each encoding proteins is not confirmed also.Due to the segmented characteristic of the genome of virus, different poison Strain between can occur again match somebody with somebody and produce height make a variation so that the strain of GCRV is complex, at present it has been reported that More than 30 separation strains, including GCRV854, GCRV861, GCRV873, GCRV875, GCRV876, GCRV991, GCRV096, GCRV829、H962、ZV-8802、ZV-8909、GCHV-854、GCRV-HZ08、JX09-01、GCRV-104、GD108、106、 109th, HuNan1307 etc., different separation strains genome sequence, genome banding pattern, cytopathy, to the side such as pathogenicity of grass carp Face differs greatly.According to existing separation strain gene sequence information, nucleotide sequence and amino acid alignment and structure are carried out Phylogenetic analysis are built, institute's toxic strain can generally be divided into 3 major classes, be divided into 3 genotype, i.e., (representative strains are the types of GCRV I GCRV-873 and GCRV-JX09-01), (representative strains are GCRV- for the types of GCRV II (representative strains are GCRV-HZ08) and the types of GCRV III 104 are).Currently, in the epidemic strain being separated in all parts of the country, three class hypotypes have been reported that, there is individually infection, also have mixing to feel Dye, is to now result in popular and outburst the main plant type of hemorrhagic disease of grass carp with the HZ08 plants of type of GCRV II as representative.

At present, for the preventing and treating of hemorrhagic disease of grass carp, also without special effective medicine and method, maximally efficient does Method is still immunoprophylaxis, the application of hemorrhagic disease of grass carp cell inactivation vaccine and cell weak-toxic vaccine, to reducing hemorrhagic disease of grass carp Serve certain effect.But the immunogenicity of inactivated vaccine is poor, it is necessary to sufficiently large amount can just cause immune Response, and it is heavy dose of can cause locally or systemically react, and the immune response for being obtained is ofer short duration, it is necessary to multiple injection To strengthen immune response, and the production cost of inactivated vaccine is relatively higher；Be present virulence and return strong danger in cell weak-toxic vaccine, have Dissipate the risk of poison.Compared with traditional vaccine, recombinant vaccine exist it is more a little its antigenic component is single, immunogenicity is strong, Body can be stimulated to produce stronger humoral and cellular immune response response, the duration is long；It is produced on a large scale, cost is relatively low It is honest and clean.Additionally, how a kind of immune effect of vaccine is, it is necessary to suitable method is detected and evaluated.Examined by immunological method The height for surveying the presence or absence of immune animal specificity antibody and antibody titer is presently the most easy and effective method.Due to grass carp Reovirus immunogenicity is poor, so far also without a kind of effective amynologic diagnostic method, is especially a lack of evaluating epidemic disease The Serology test of seedling immune effect.

The content of the invention

Research has shown that：The type S9 gene segments of GCRV II can encode one up to 418 protein of amino acid (VP6), predicted molecular weight is 42.86KDa, and the result of BLASTp shows the VP6 albumen and Aquareovirus VP6 albumen (S8 codings), mammal (MRV) σ 2 (S2 codings), Avianreovirus (ARV) σ A (S2 codings) have homology higher, With U.S.'s GCRV (AGCRV), 873 plants of GCRV (GCRV873), Aquareovirus A (Aqu A), the homology of America gold body bream reovirus (GSV), MRV serotypes 2, MRV serotypes 3 and MRV serotypes 1 is respectively： 25%th, 26%, 25%, 26%, 20%, 20% and 20%, similitude is respectively：37%th, 39%, 37%, 40%, 36%, 36% and 37%, it is about 24% and 38% with the homology and similitude of ARV.It was initially believed that the encoding proteins VP6 of HZ08S9 There is close affiliation with the σ A albumen of the σ 2 and ARV of MRV.GCRV II is the virus of new separation and research in recent years, to disease The protein function of each sections coding of virus gene group is not clear, and which is structural proteins, and which is also not fixed non-structural protein Whether it is structural proteins always also without it is experimentally confirmed that also not knowing the concrete function of the albumen by the albumen of, S9 gene codes With immunogenicity how.

We are verified by experiments, and the VP6 albumen of the type S9 gene codes of GCRV II is structural proteins, and immune animal can be induced to produce The specific antibody of raw more efficient valency, with good immunogenicity；Antibody for VP6 albumen can be with specific recognition GCRV II type strain, and there is very strong neutralising capacity to the type strain；Further experiment shows, with the immune grass of VP6 recombinant proteins Fish, can induce grass carp and produce specific antibody higher, and the attack of GCRV velogen strain can be played necessarily Immanoprotection action.Therefore, to the research and application of the type VP6 albumen of GCRV II, split hair hemorrhagic disease of grass carp new generation vaccine and Immunological detecting kit is extremely important.

Thus, it is an object of the present invention to provide a kind of VP6 antigen proteins, and the Anti-TNF-α prepared by it Body.

It is another object of the present invention to provide above-mentioned VP6 antigen proteins and the polyclonal antibody of anti-VP6 antigen proteins Application in preparing hemorrhagic disease of grass carp recombinant vaccine or preparing in GCRV detection kit.

The technical solution used in the present invention is：

A kind of type VP6 albumen of GCRV II, its amino acid sequence such as SEQ ID NO:Shown in 2.

A kind of nucleic acid for encoding albumen described in claim 1, its base sequence such as SEQ ID NO:Shown in 1.

Above-mentioned VP6 albumen as fish reovirus antigen protein application.

A kind of polyclonal antibody, it with amino acid sequence is SEQ ID NO to be:Albumen shown in 2 is antigen, and animal system is immunized Standby gained.

The polyclonal antibody of above-mentioned VP6 albumen and/or above-mentioned anti-VP6 albumen is preparing hemorrhagic disease of grass carp genetic engineering epidemic disease Application in seedling.

The polyclonal antibody of above-mentioned VP6 albumen and/or above-mentioned anti-VP6 albumen is preparing GCRV detection examination Application on agent box.

A kind of recombinant vaccine, the polyclonal antibody containing above-mentioned VP6 albumen or anti-VP6 albumen.

A kind of kit for detecting GCRV, contains above-mentioned VP6 albumen and/or above-mentioned anti-VP6 albumen Polyclonal antibody.

The beneficial effects of the invention are as follows：

The recombinant protein that the present invention is obtained has preferable immunogenicity, compared with GCRV other structures albumen, its induction The specific antibody titres that immune animal produces are higher, and its antibody titer can reach 1：:, 2048, and other structures albumen is lured The specific antibody titres led are general 1:Less than 1024,.Therefore, the VP6 albumen of S9 gene codes is to develop hemorrhagic disease of grass carp The preferable candidate antigens of recombinant vaccine.Using VP6 albumen as detection GCRV antibody titer it is indirect enzyme-linked The envelope antigen of immunosorbent adsorption test (ELISA), can well recognize the specificity that GCRV infection or vaccine immunity grass carp produce Antibody；Also, the envelope antigen obtained by the method, has the advantages that quick, special, sensitive, easy and safe, overcomes Tissue and cell cultivation method produces that the operating process that is caused as indirect ELISA envelope antigen of intact virus is cumbersome, shakiness Determine, yield poorly, the shortcoming of high cost.Therefore, the type S9 genes of GCRV of the invention II and its encoding proteins, split hair grass carp hemorrhage Sick new generation vaccine, immunological detecting kit and treatment product have great importance, for hemorrhagic disease of grass carp provides new effective Solution route.

Brief description of the drawings

Fig. 1 is the type strain S9 gene PCR amplified production electrophoretograms of GCRV II, and M is DL marker 1000 in Fig. 1；1 is the moon Property control；2 is the pcr amplification product with GCRV HZ08 strain virus cDNA as template；

Fig. 2 is recombinant plasmid pET-32a-S9 double digestion qualification results, and M is DL marker 15000 in Fig. 2；1 is restructuring Plasmid pET-32a-0 double digestion products；

Fig. 3 is that SDS-PAGE analyzes recombinant plasmid pET-32a-S9 expression products and its solubility, and M is low molecule in Fig. 3 Quality protein marker；1 is the thalline of the pET-32a (+) of IPTG inductions；2 is the pET-32a-S9 bacterium without IPTG inductions Body；3 is the supernatant after thalline ultrasonic disruption；4 is the pET-32a-S9 thalline induced through IPTG；5 is thalline ultrasonic disruption Precipitation afterwards；

Fig. 4 is S9 gene codes recombinant protein through Ni posts SDS-PAGE analysis results, M low molecules quality in Fig. 4 after purification Protein marker；1 is unpurified bacterial protein；2 is the VP6 recombinant proteins of purifying；3 is to flow through liquid by Ni posts；

Fig. 5 is that western blot analyze VP6 protein antiserum characteristics, M Low molecular weight proteins marker in Fig. 5；1 It is VP6 recombinant proteins；2 is the HZ08 strains of purifying；

Fig. 6 is indirect immunofluorescence analysis VP6 protein antiserum characteristics, and A is the GSB cells of HZ08 plants of inoculation in Fig. 6；B It is the normal GSB cell controls of non-virus inoculation.

Specific embodiment

With reference to embodiment, the present invention is further illustrated, but is not limited thereto.

Molecular biology experiment technology employed in following examples include PCR amplifications, plasmid extraction, plasmid convert, DNA fragmentation connection, digestion, gel electrophoresis etc., unless otherwise specified, generally conventionally operate, and for details, reference can be made to《Molecule Cloning experimentation guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated, 2002, Beijing：Science Press), or according to the condition proposed by manufacturer.

Embodiment

First, the extraction of GCRV HZ08 plants of RNA and S9 gene fragment amplifications

(Qiagen companies, article No. are purchased from according to Qiagen RNA extracts kits：74104) specification step, from sense Middle extraction RNA in the CIK cell of GCRV HZ08 strain virus is contaminated, with TAKARA (purchased from Dalian treasured bioengineering Co., Ltd, goods Number：) reverse transcription reagent box is by reverse transcription of viral RNA into cDNA.GCRV HZ08 Strain is protected by Zhujiang River aquatic products research institute's fish disease room Hide.

According to GenBank (GenBank:GU350746.1 GCRV-HZ08 plants of S9 gene order designs 1 pair of primer in), on Trip primer：5’-GATGGA TCCATA GTC TCG GAA GCC TAC CAA-3’(SEQ ID NO:3)；Anti-sense primer： 5’-ATA CTC GAGAGC ACG CGT CCA GTT ATT-3’(SEQ ID NO:4), in upstream and downstream primer sequence respectively Insertion BanH I and the restriction enzyme sites of Xho I (gene with underscore), the cDNA with above-mentioned preparation are carried out as masterplate to S9 genes PCR is expanded.2.5 μ L 10 × PCR reaction buffer, 0.5 μ L dNTP (10mM are added in every 25 μ L reaction systems Each), upstream and downstream primer is respectively 0.5 μ L, 2 μ L cDNA as masterplate, LA Taq enzymes 0.25 μ L, ddH₂O 18.75μL；Instead The condition is answered to be：95 DEG C of predegenerations 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, totally 35 circulations, 72 DEG C of extension 8min.

PCR primer detects that it is of the same size that an expection occurs at about 750bp in discovery through 1% agarose gel electrophoresis Specific amplification band (see Fig. 1), with glue reclaim kit recovery purifying purpose band, is consistent through sequence verification with expection.

2nd, pET-32a-S9 recombinant expression carriers build

By PCR recovery products and prokaryotic expression carrier pET-32a (+) respectively double digestion treatment (the I 1 μ l of μ l, Xho of BanH I 1, 10 × H buffer 2 μ l, μ l of plasmid 16) after, through 1% agarose electrophoretic analysis, glue reclaim S9 purpose fragments and handle well PET-32a (+) carrier, with T4DNA ligases connect S9 purpose fragments and pET-32a (+) (the μ l of T4Liqase 1.5, The μ l of 1.5 μ l, pET-32a (+) of T4Liqase buffer, 3 μ l, S9 purpose fragments 9, totally 15 μ l), connection product is transformed into greatly Enterobacteria DH5 α, screening positive clone, bacterium solution PCR be accredited as the positive extraction plasmid, plasmid through double digestion and sequencing identification just After really, pET-32a-S9 is named as, preserved for follow-up test.

It is correct that PCR and double digestion qualification result (see Fig. 2) show that recombinant expression plasmid pET-32a-S9 builds, sequencing knot Fruit confirm purpose fragment and reading frame it is correct, and genes of interest S9 and pET-32a (+) His-Tag label proteins same Under ORFs.

3rd, induced expression and soluble analysis

Induced expression：To identify that correct recombinant plasmid pET-32a-S9 is transformed into e. coli bl21 (DE3), picking The positive colony of LB/Amp plate screenings, in LB/Amp culture mediums, 37 DEG C, 200rpm/min constant-temperature tables culture to optical density (OD600=0.6) when, the TPTG inductions of final concentration of 1mmol/L are added, taking 200 μ l bacterium solutions after induction 10h is collected by centrifugation thalline It is standby with supernatant, while setting up empty carrier pET-32a (+) as negative control.

Soluble analysis：By pET-32a-S9 induced expression 20ml, thalline is collected by centrifugation, with resuspended centrifuge washings of PBS 2 times Afterwards, the 5ml resuspended thalline of PBS are added, it is clear to solution in ultrasonic disruption on ice (work 5s, interval 10s, power 250w) about 2h Clearly, the broken liquid of 200 μ L is drawn, supernatant is drawn onto another EP pipes and is designated as solution A by 6000rpm/min centrifugation 5min, and precipitation uses 200 μ The urea dissolving of l 6M, is designated as solution B, and A, B solution are carried out into SDS-PAGE respectively, and soluble point is carried out to the recombinant protein Analysis, if destination protein is main in solution A, the albumen is soluble protein；If destination protein is main in B solution, mesh Albumen with inclusion bodies exist.

Show that recombinant plasmid pET-32a-S9 bacterial strains go out at 42ku through the recombinant protein of SDS-PAGE analysis induced expressions An existing purpose band (see Fig. 3), the His- label proteins of pET-32a (+) carrier amalgamation and expression together with destination protein.Take The solution B of supernatant solution A and 6M the urea dissolution precipitation after the centrifugation of ultrasonic disruption liquid, through SDS-PAGE analysis shows, fusion The destination protein of label protein is primarily present in B solution, i.e., destination protein is main exists (see Fig. 3) with inclusion bodies.

4th, the optimization of induced expression condition

Inductive condition is optimized using chessboard method, pET-32a-S9 is transformed into e. coli bl21 (DE3) competence, LB/Amp plate screenings positive bacterium colony, picking single bacterium is fallen within 3ml LB/Amp culture mediums, and at 37 DEG C, 200rpm/min constant temperature shakes , then be inoculated with bacterium solution in 20 μ L to 2mL LB/Amp culture mediums by bed culture 12h, in 37 DEG C, the training of 200rpm/min constant-temperature tables Support during to optical density (OD600 setting 0.2,0.4,0.6,0.8 and 1.0, and practical measurement is defined), add TPTG induction (IPTG ends Concentration sets 0.5,1.0,1.5,2.0,3.0mmol/L), the centrifugation of 200 μ l bacterium solutions, collects thalline, through SDS-PAGE are taken after culture 10h With the relative expression quantity of the software analysis recombinant proteins of BandScan 5.0 after analysis.

Finally draw when induced expression condition is：When OD600 is 1.0 or so, IPTG final concentration of 1.6mmol/L, its table Optimal up to condition, the content of destination protein is up to more than 70%.

5th, the great expression of destination protein and purifying

Picking LB/Amp plate screenings obtain positive single bacterium and fall 1mL LB/Amp 10~12h of medium culture, then by it It is inoculated into 1L LB/Amp culture mediums, 37 DEG C, 200rpm/min constant-temperature table cultures, the induced expression under the expression condition of optimization. The bacterium solution of induced expression is centrifuged 5min in 10000rpm/min, collects thalline, with resuspended centrifuge washings of 400ml PBS 2 times, then Add 100ml PBS resuspended, clarified in ultrasonic disruption on ice to solution.Broken liquid is collected by centrifugation into inclusion body, and (albumen can Dissolubility verifies as inclusion body protein), purify purpose referring next to Ni- gel purification kits (inclusion body protein purifying) specification Albumen.Inclusion body is dissolved with 50ml Binding Buffer overnight, lysate 10000rpm/min is centrifuged in 20min collections Clearly, then 0.45 μm of membrane filtration, supernatant is loaded the 10 times of column volume/hours of Ni column flow rates for having balanced, collection is flowed through Liquid；Uncombined albumen and foreign protein are washed away with the Binding Buffer of 15 times of column volumes, 20ml Elution are then used Buffer elutes destination protein, collects eluting peak (whole process of purification is carried out in 4 DEG C of refrigerators).Egg under being eluted on Ni posts It is white to load bag filter, it is sequentially placed into containing 6M, dialysed in the PBS solution of 4M, 2M, 1M, 0.5M, 0M urea, each concentration dialysis 12h (all processes are carried out in 4 DEG C of refrigerators).PEG-6000,4 DEG C of refrigerators is added to stand concentration, divide after concentration 50% after the completion of dialysis 1mL/ pipes are dressed up, is saved backup with -20 DEG C after UV absorptiometry survey concentration.

6th, the preparation of polyclonal antibody

Kunming small white mouse is injected after the destination protein of purifying is diluted into 200 μ g/ml with aseptic PBS.It is immune for the first time to use Freund's complete adjuvant 1:1 emulsification, injection volume is 200 μ L/, and 10 are immunized altogether.Second immune and immune Freund of third time Freund's incomplete adjuvant 1:1 emulsification, injects same amount.Each immunization time interval 14d.Dock the immune 10d day after tomorrow for the third time and take blood, Serum is prepared, the potency of serum antibody is detected with ELISA.Such as antibody titer not enough, can be such as sufficiently high with booster immunization once, Then 14d plucks eyeball and takes blood.4 DEG C of refrigerators stand overnight after 37 DEG C of the blood sample taken stands 1h, 3000rpm/min centrifugations in second day 8min, collects serum, and -20 DEG C save backup.Control group injects aseptic PBS and prepares negative serum.

7th, serum antibody titer is determined

Serum antibody titer is determined according to the GCRV indirect ELISA detection method that this laboratory is set up, The indirect ELISA method set up as antigen coat polystyrene reactant plate with the VP6 albumen of purifying.Its simplified process is such as Under：The recombinant protein that will be purified presses 1 with the carbonate buffer solution of pH9.6:10000 times of dilutions (50ng/ml) are coated with 100 μ L in 96 Hole elisa Plates, 4 DEG C overnight；With 200 μ L 1%BSA 2h is closed in 37 DEG C；The serum that will be prepared presses 1:10¹、1:10²、1:10³、1: 10⁴、1:10⁵、1:10⁶、1:10⁷、1:10⁸、1:10⁹、1:10¹⁰Constant gradient dilution adds 100 μ L, 37 DEG C of incubation 2h, with corresponding dilute The negative serum for releasing multiple is compareed；Add 1：Sheep anti-mouse igg 100 the μ L, 37 DEG C of incubation 1h of the HRP marks of 5000 dilutions；With On after often step reaction all with 200 μ L PBST board-washings 3-5 times, each 5min；Finally with TMB colour reagents box colour developing 20min, plus Enter the H2SO4 color development stoppings of 0.5M, OD450 values are read on ELIASA (Infinite M200PRO).When OD450 >=0.277, and P/N >=2.1 are judged to the positive.

Titration is carried out to VP6 recombinant proteins Mouse Antisera with indirect elisa method, as a result shows that the antibody titer is about 1:10⁵。

8th, polyclonal antibody specificity analysis

Western blot are analyzed：GCRV HZ08 viruses, pET-32a (+) empty carrier and pure of IPTG inductions that will be purified The VP6 recombinant proteins of change through SDS-PAGE electrophoresis, electrophoresis terminate after with half-dried transferring film method by protein delivery to NC films, with containing 4 DEG C of closings of TBST buffer solutions of 5% skim milk are overnight；Add 1:The serum of 1000 dilutions, 37 DEG C of incubation 2h；Add 1:5000 The sheep anti-mouse igg of the HRP marks of dilution, 37 DEG C of incubation 1h；Washed 3~5 times with TBST after per secondary response, each 5min is finally used DAB chromogenic reagents simultaneously observe result.

The polyclonal antibody of preparation is analyzed through western blot, in the duct of the GCRV HZ08 strains of addition purifying, Occur a specific band at about 42ku, it is in the same size with the albumen that viral VP6 itself is encoded；Adding the restructuring egg of purifying In white duct, occurs a specific band at about 40ku, with expected size always；And in adding the duct of empty carrier thalline There is no any band.

Indirect immunofluorescence assay (Indirect immunofluorescent assay, IFA)：By GSB passages Into 96 porocyte culture plates, after cell is long be paved with 80% or so to individual layer after, infection HZ08 virus；After infection virus 5 d, will Culture medium in Tissue Culture Plate is exhausted, and is washed with the PBS of 0.01mol/L 3 times, adds 80% acetone soln incubation at room temperature 30min, sucks acetone, drying at room temperature 1h；Add 100 μ l 1 per hole：The anti-VP6 protein polyclone antibodies of mouse of 1000 dilutions, 37 DEG C 1h is incubated, PBST is washed 3 times, adds 1:The μ l of fluorescence secondary antibody 100 of the sheep anti-mouse igg-FITC marks of 50 dilutions, 37 DEG C of incubations 1h；PBST is washed 3 times, and result is observed under fluorescence inverted microscope (Nikon, Eclipse Ti-S).Negative control is to be uninfected by The normal GSB cells of virus.

The result of indirect immunofluorescence experiment shows that the Anti-TNF-α physical efficiency of preparation makes the GSB of infection HuNan1307 strains Cell can produce specific fluorescence (see A in Fig. 6), and fluorescence letter is not observed in the normal GSB cells of uninfecting virus Number.

Above example is only to introduce preferred case of the invention, to those skilled in the art, without departing substantially from this Any obvious changes and improvements carried out in the range of spirit, are regarded as a part of the invention.

SEQUENCE LISTING

<110>China's Pearl River Fishery Research Institute of Aquatic Science Research Institute

<120>The recombinant protein of the type S9 gene codes of GCRV II and its application

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Claims

1. a kind of VP6 albumen of the type S9 gene codes of GCRV II, its amino acid sequence such as SEQ ID NO:2 institutes Show.

2. it is a kind of encode claim 1 described in albumen gene, its nucleotide sequence such as SEQ ID NO:Shown in 1.

3. the VP6 albumen described in claim 1 as GCRV antigen protein application.

4. a kind of polyclonal antibody, it is characterised in that it with amino acid sequence is SEQ ID NO to be:Albumen shown in 2 is antigen, Immune animal prepares gained.

5. the polyclonal antibody described in the VP6 albumen and/or claim 4 described in claim 1 is preparing hemorrhagic disease of grass carp base Because of the application in engineered vaccine.

6. the polyclonal antibody described in the VP6 albumen and/or claim 4 described in claim 1 exhales the lonely disease of intestines in preparation grass carp Application in malicious detection kit.

7. a kind of recombinant vaccine, it is characterised in that containing the VP6 albumen described in claim 1 or contain claim 4 institute The polyclonal antibody stated.

8. it is a kind of detect GCRV kit, it is characterised in that containing the VP6 albumen described in claim 1 and/ Or the polyclonal antibody described in claim 4.