CN102532310B - Monoclonal antibody against grass carp reovirus VP5 protein and application of monoclonal antibody - Google Patents
Monoclonal antibody against grass carp reovirus VP5 protein and application of monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody against grass carp reovirus VP6 protein and application of the monoclonal antibody. The monoclonal antibody against the grass carp reovirus VP6 protein is secreted by a mouse hybridoma cell strain with the collection number of CGMCCNo.5796. The monoclonal antibody against the grass carp reovirus VP6 protein can be used for detecting grass carp reovirus. The invention also discloses an enzyme-linked immuno sorbent assay (ELISA) kit for detecting the grass carp reovirus.
Description
Technical field
The invention belongs to field of immunology, be specifically related to a kind of monoclonal antibody of anti-GCRV VP5 albumen, hybridoma cell strain and set up a kind of sandwich ELISA method that detects GCRV.
Background technology
Hemorrhagic disease of grass carp is the fish disease caused by virus of a kind of hyperinfection, lethality, is popular in culture zone, China various places, and especially general with the Yangtze valley and Guangdong, Guangxi, Fujian, mortality ratio has a strong impact on the development of China's grass carp aquaculture up to 80%.Its cause of disease is GCRV (Grass carp reovirus, GCRV), is the first strain Aquatic animals virus (Chen Yanxin, Jiang Yulin of isolated in China, evaluation.Science Bulletin, 1983,28:1138-1140).The reports such as Meyers TR in 1979 are isolated the first strain and are exhaled the lonely sample viruses of intestines (Meyers T R. J Gen Virol, 1979,43:203 ~ 212) from hydrocoles.At present in the world isolation identification more than 40 plant Aquareovirus (aquareovirus).Yet in these isolation identification strain out, most of strains can not cause host's pathologic reaction or only show weak pathogenic.GCRV is the strongest strain of virulence in the Aquareovirus.
The genome of GCRV is comprised of 11 dsRNA fragments, and virus genomic total molecular weight difference little (about 15 * 10 between different strains
6Da), and each fragment molecular weight difference to some extent, press molecular size range, 11 fragments can be divided into 3 groups, namely (wait quietly in the field than large fragment (L1, L2, L3), medium fragment (M4, M 5, M6) with than small segment (S7, S8, S9, S10, S11), China's virusology, 1999,4 (1): 87-92; Fang Min, Huang Hualiang. biotechnology journal, 2001,17 (6): 608-612).Ripe GCRV particle is comprised of 7 kinds of structural protein and 4 kinds of Nonstructural Proteins, and wherein viral protein VP5 and VP7 have consisted of viral outer capsid component.Disclosed VP5 and VP7 plays irreplaceable effect at virus infection and in causing a disease by protease cracking experiment.
China is a big country of cyprinid fish cultivation.When some country of outlet, the inspection and quarantine unit of import and export is to detect to GCRV.Making a definite diagnosis of this disease can only be attempted at present with finishing with methods such as PCR or direct electrophoresis after the cellular segregation virus.Mainly be that immunological detection method also is not very ripe owing to there be not antigen and antiserum(antisera) well.The antiserum(antisera) that the import and export inspection and quarantine of China obtains or detection method be in the urgent need to.Acquisition has the monoclonal antibody of the stronger anti-GCRV of specificity, not only has quick, sensitive characteristics, but also applicable large-scale the detection.Monoclonal antibody technique at home and abroad has been a kind of very proven technique, yet there is not yet the relevant report of the monoclonal antibody with the stronger anti-grass carp arc intestines arc virus of specificity.
Summary of the invention
First technical problem to be solved by this invention provides a kind of monoclonal antibody of anti-GCRV VP5 albumen.
Second technical problem to be solved by this invention provides a kind of ELISA test kit that detects GCRV.
The 3rd technical problem to be solved by this invention provided a kind of method of vitro detection GCRV.
The 4th technical problem to be solved by this invention provided a kind of application of monoclonal antibody in detecting GCRV of anti-GCRV VP5 albumen.
A kind of virus contains plurality of antigens, and a kind of antigen may contain a plurality of antigen sites again.Therefore can obtain more than a kind of antibody for a kind of antigen, but these antibody may be different to the binding characteristic of antigen.In order to find the monoclonal antibody that to be combined with antigen-specific, need to carry out in a large number repeatedly comparison, screening and identification work.The present invention has obtained to secrete the hybridoma cell strain of specific binding grass carp arc length virus VP 5 albumen by a large amount of work; And on this basis, set up first and detected the sandwich ELISA detection method of GCRV, and prepared the ELISA test kit that detects GCRV, and can be used for the detection of extensive GCRV, have very wide application prospect.
For solving the problems of the technologies described above, technical scheme provided by the present invention is as follows:
The monoclonal antibody of anti-GCRV VP5 albumen provided by the present invention is to be the mouse hybridoma cell strain secretion generation of CGMCC No.5796 by deposit number.This mouse hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 27th, 2012 and (is called for short CGMCC, the address is No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city), deposit number is CGMCC No.5796 for its deposit number.
Deposit number is that the mouse hybridoma cell strain of CGMCC No.5796 also belongs to protection scope of the present invention.
The present invention also provides a kind of ELISA test kit that detects GCRV, and this test kit comprises monoclonal antibody of the present invention.Further, also comprise the solid phase carrier of the polyclonal antibody of coated anti-GCRV in this test kit, the antibody of the anti-mouse of enzyme mark.Described enzyme is preferably horseradish peroxidase.Adopting the antibody of the anti-mouse of enzyme mark of commercialization purchase, is because on the one hand, if the monoclonal antibody of explaining among mark the present invention or polyclonal antibody, the process more complicated.Even success all may affect tiring or other aspects of antibody of antibody, make whole test kit research and development more loaded down with trivial details, consumption power, consuming time.And use the antibody of the anti-mouse of commercialization enzyme mark, then there is not this problem; On the other hand, if the monoclonal antibody among mark the present invention or polyclonal antibody, the cost of whole test kit is just very high, and it is infeasible applying for test kit.Use the antibody (sigma) of the anti-mouse of commercialization enzyme mark, every 1ml expense is about 2000 yuan, has reduced the cost of test kit.
Further, for the ease of detecting, test kit of the present invention also comprises positive control and negative control, and the required enzyme linked immunosorbent detection reagent of ELISA reaction.Wherein, described positive control is GCRV solution, and negative control is fish cell suspension or healthy tissues homogenate.Described enzyme linked immunosorbent detection reagent is conventional enzyme linked immunosorbent detection reagent, includes but not limited to substrate reactions liquid, washings and the reaction terminating liquid of enzyme.
The preparation process of the polyclonal antibody of anti-GCRV of the present invention is as follows: with the GCRV of gradient centrifugation purification as antigen, the multi-point injection immune goat, be respectively per 1 all immunity once, immunity is 4 times altogether; Get the polyclonal antibody that the serum purifying is anti-GCRV; Extracting IgG through the supersaturated ammonium sulfate method of purification, as the first antibody in the detection method.
The present invention also provides a kind of method of vitro detection GCRV, and the method may further comprise the steps:
(1) with the testing sample application of sample in the solid phase carrier that is coated with first antibody, thereby GCRV in the testing sample and the first resistive connection on the solid phase carrier are closed, form the solid phase carrier with " first antibody-virus " binary complex; Described first antibody is the polyclonal antibody of anti-GCRV.
(2) solid phase carrier that the second antibody application of sample is obtained in (1), thus formation is with the solid phase carrier of " first anti--virus-second antibody " three elementary lengths and nothing; Described second antibody is monoclonal antibody of the present invention.
(3) solid phase carrier that the antibody application of sample of the anti-mouse of enzyme mark is obtained in (2), thus form with " antibody of first antibody-virus-second antibody-anti-mouse of enzyme mark " tetraplex.
(4) detect enzyme labelling thing in the tetraplex, the existence of determining GCRV in the testing sample whether or the amount that exists, thereby the existence of determining GCRV whether.
The present invention has the following advantages and effect
1. obtain the monoclonal antibody of anti-GCRV VP5 albumen and secrete this monoclonal antibody hybridoma cell strain.Can prepare a large amount of required specific antibodies after this hybridoma proliferation; Behind the hybridoma injection mouse, the ascites MAb mediated ELISA of generation is tired and is 1:1.6 * 10
5Hybridoma cell strain is active high, in the liquid nitrogen after frozen 8-10 month, and still can rapid fluid resuscitation and keep excellent activity.In the preparation of described monoclonal antibody, cell confluency is 95.6%, and positive rate is 97.0%.
2. the antigen of immune mouse is by according to the VP5 gene order of GCRV design primer, pcr amplification be about fragment about 2000bp, use albumen about artificial 77 KDa that express of prokaryotic expression method as immunizing antigen.Find that by scholar's research ripe GCRV particle is comprised of 7 kinds of structural protein and 4 kinds of Nonstructural Proteins, wherein viral protein VP5 and VP7 have consisted of viral outer capsid component.Disclosed VP5 and VP7 plays irreplaceable effect at virus infection and in causing a disease by protease cracking experiment.VP5 also can carry out the change of conformation in addition, and its effect is to guarantee that film penetrates.VP5 albumen is the outer capsid albumen of GCRV, may have higher immunogenicity.The albumen size of expressing has very important relation with immunogenicity, more large easier generation immunogenicity, otherwise a little less than.But the molecular weight of albumen that anticipation is expressed is larger, and manually expressing difficulty also increases accordingly.The such design of the present invention is from comprehensive angle, has the advantage of four aspects: one, avoided the problem of GCRV purification difficulty; Its two, antigen has certain specificity, has alleviated the extensive work amount that screening brings in the anti-GCRV monoclonal antibody for preparing; Its three, highly purified albumen has strengthened immune effect as antigen; Last aspect, the molecular weight of albumen that we select is larger, and immunogenicity is more intense.Positive effect after the assurance cytogamy.
3. in screening process, used indirect ELISA method, the embedding plank is taked the measure aspect two in this method, and a large amount of elisa plates that have been disposable embeddings on the one hand guarantee the unity of whole experiment; On the other hand, in the embedding sample, except normal negative control (cell suspension), GCRV, blank, also embedding is as the recombinant protein of immunogenic artificial expression.The so dual accuracy that has guaranteed to obtain the monoclonal antibody of the anti-GCRV of preparation of purpose, the impact of having avoided some modifications of artificial expression recombinant protein to bring.
4. the sandwich ELISA method of the detection GCRV of setting up can detect viral whether existence the in the viral and tissue in the cell suspension accurately.This detection method is providing favourable condition for importing and exporting inspection and quarantine mechanism aspect the inspection and quarantine GCRV.
Description of drawings
Fig. 1. the enzyme of manually expressing the recombinant plasmid pET30 α-gv-vp5 of GCRV protein part is cut and the PCR evaluation: (A) M:DNA maker; 1-2, GCRV virus VP 5 gene RT-PCR amplification 3, blank; (B) M:DNA maker; 1-2, recombinant plasmid SacI, XhoI double digestion 3, recombinant plasmid PCR 4, recombinant plasmid.
Fig. 2. manually express the washing of expression, inclusion body and the purifying of the recombinant protein of GCRV protein part: (A) M: molecular weight of albumen standard, 1, ultrasonic postprecipitation 2, ultrasonic rear supernatant 3, washing primary sedimentation 4, supernatant 5 of washing, washed twice precipitation 6, washed twice supernatant 7, three precipitations 8 of washing, three supernatants 9 of washing, do not induce recombinant plasmid;
M: recombinant protein behind molecular weight of albumen standard 1-2, the purifying.
Fig. 3. recombinant protein Western blotting detects behind the purifying: (A) M: recombinant protein 2 behind molecular weight of albumen standard 1, the purifying, do not induce pET30 α-gv-vp5 whole bacterial protein 3, pET30 α whole bacterial protein; (B) M: molecular weight of albumen 1, goat-anti GCRV antiserum(antisera) are antibody 2, normal sheep serum negative control.
Fig. 4. M: VP5 reaction result in VP5 reaction result 4, monoclonal antibody and artificial expression the in SDS-PAGE 3, monoclonal antibody and the virus of the SDS-PAGE 2 of molecular weight of albumen 1, GCRV, the artificial VP5 that expresses.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The preparation of the monoclonal antibody of embodiment 1 anti-GCRV VP5 albumen
1. the preparation of antigen
According to GCRV (Grass carp reovirus)---and sequence that the gv-vp5 gene of GCRV is delivered at GenBank (accession number: AF239175), design primer GV-U and GV-D.Upstream primer is introduced the SacI site, and downstream primer is introduced the XhoI site.And synthetic by giving birth to worker biotech firm.
The RNA that the present invention extracts take the GCRV viral suspension is as template, with
5 '-
GAGCTCGG ACTTCGCACTCTCTCTACAATG-3 ' and
5 '-
CTCGAGAA GTTCACGCGGGCATGGAAG-3 ' is primer, by RT-PCR amplification obtain the encoding goal gene of VP5 albumen.The gene that obtains is connected to the pGEM-T-vector carrier, does not contain sudden change through the gene sequencing checking.Add the primer of SacI and XhoI restriction enzyme site by two ends, above-mentioned clone's gene is connected to pET-30 α carrier (available from Novagen company), obtain p ET-GV-VP5 recombinant plasmid.This recombinant plasmid transformed is arrived in BL21 (DE3) the expression type intestinal bacteria again, induce under the IPTG condition, the albumen of expression mainly is present in the precipitation.The His-tag label purifying that VP5 albumen in the precipitation of expressing carries by carrier, purifying adopts Ni-NTA HisBind resin purification (MERCK company).The protein concentration 0.6mg/ml of purifying is total to 200ml, 120mg.Expression after VP5 protein induced expression front and back and enzyme are cut is seen Fig. 1.Before the VP5 albumen pronucleus expression was induced, the expression before and after the ultrasonic disruption cell was seen Fig. 2.Recombinant protein behind the purifying is carried out Western blotting to be detected, the result is as shown in Fig. 3: be antibody with the Histag monoclonal antibody, one specific reaction band (seeing figure A) appears at nitrocellulose filter 77kDa place, simultaneously take goat-anti GCRV antiserum(antisera) as antibody, the specific reaction band (seeing figure B) that same molecular amount size also can occur has confirmed that the 77kDa band is for containing the fusion expressed product of target protein.
A kind of optimal way of the present invention, described expression vector are the pET-30a carrier, and wherein with 6 * His label, this tag molecule amount is very little, can be directly used in later experiments behind the purifying.Expression vector is introduced in the genetically engineered host cell, can be expressed described albumen.In this experiment, adopting host cell is intestinal bacteria (DE3).
The method of prokaryotic expression hemorrhagic disease of grass carp coat protein VP5 is adopted in this experiment, efficiently expression in escherichia coli and purifying VP5 albumen, expression amount is high, high specificity, being easy to purifying and later stage carries out protein renaturation and makes it have complete space conformation.
2. immune mouse
The mouse of immunity usefulness is SPF level female BALB/c mouse in 5 age in week.The artificial expression grass carp arc intestines arc virus VP 5 albumen of every mouse after with above-mentioned purifying carries out the abdominal injection fundamental immunity as antigen, manually expresses recombinant protein (60 microgram) and mixes abdominal injection with Freund's complete adjuvant 1:1; Booster immunization after 2 weeks is manually expressed recombinant protein (60 microgram) and is mixed abdominal injection with Freund's incomplete adjuvant 1:1; Afterwards every 1 all booster immunizations 1 time, abdominal injection is manually expressed recombinant protein and is injected 120 micrograms/only; After the 4th immunity the 3rd day, mouse is taken off cervical vertebra put to death, the aseptic spleen of getting is used for cytogamy.
3. cytogamy
Conventional cell-fusion techniques: get after the spleen of immune mouse and SP2/0 myeloma cell merge, the thymocyte that adds mouse is cultivated altogether cultivation in the system with fused cell at HAT.
The preparation of the HAT substratum of using in the described technology: with 50 times of concentrated HAT(2ml, GIBCO company) and superfine foetal calf serum (20ml, Hangzhou Sijiqing Biological Engineering Material Co., Ltd.) joins mixing in the modified form RPMI1640 substratum (80ml, hyclone company).
4. the screening of hybridoma and clone
Fused cell was cultivated after 10 days, collected cells and supernatant, carried out the indirect ELISA detection with the GCRV of gradient centrifugation purification and the GCRV VP5 albumen of artificial expression as antigen.The positive hybridoma cell strain limiting dilution assay that filters out is cloned.Wherein positive mouse hybridoma cell strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (abbreviation CGMCC on February 27th, 2012, the address is No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city), deposit number is CGMCC No.5796.
5. ascites induces
Get female Balb/C mouse in 6～8 ages in week, aseptic whiteruss 0.5 ml of intraperitoneal injection/only; After 1 week, the intraperitoneal injection positive hybridoma cell; The inoculation hybridoma after 7～10 days, see that mouse web portion obviously expands, tap the abdomen, centrifugal rear collection supernatant ,-80 ℃ frozen for subsequent use.
Embodiment 2: the CHARACTERISTICS IDENTIFICATION of the monoclonal antibody of anti-GCRV VP5 albumen
1. the titer of ascites of monoclonal antibody is identified
Method: adopt indirect elisa method that the titer of ascites of monoclonal antibody is identified.
The result: monoclonal antibody titer of ascites of the present invention is 1:1.6 * 10
5, show that hybridoma cell strain has the ability of the high titre antibody of secretion.
2. the hypotype of monoclonal antibody is identified
Method: use the parting kit (SBA Clonotyping System/HRP) with the SouthernBiotech of the U.S., according to its specification sheets the Ig hypotype of monoclonal antibody of the present invention is identified.
The result: the hypotype of monoclonal antibody of the present invention is IgG2b type k chain.
3. the specificity analyses of monoclonal antibody
Immunoblotting (western blotting):
Take purifying GCRV and artificial albumen of expressing as test set, if normal EPC cell lysate is opposite sex contrast, the positive contrast of immune serum, through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein isolate, electricity consumption turns method the albumen on the gel is transferred to respectively nitrocellulose filter (NC film, aperture 0.20 μ m) on, 4 ℃ of sealings are spent the night in containing the 0.01MPBS solution that quality volume percentage is 10% skim-milk; PBS-T(contains in the PBS solution that volume percent is 0.05% Tween-20) after the washing, add the ascites (monoclonal antibody 1:1000 is diluted in PBS) of mouse, hatch 1h for 37 ℃; PBS-T(contains in the PBS solution that volume percent is 0.05% Tween-20 again) after the washing, take the goat anti-mouse igg of horseradish peroxidase (HRP) mark as two anti-, hatch 1h for 37 ℃; After the washing, observe with substrate mixed solution NBT/BCIP colour developing.
The result: see Fig. 4, the monoclonal antibody of the anti-GCRV of the present invention preparation, can be special with virus in VP5 albumen and artificial VP5 albumen of expressing react.Artificial VP5 molecular weight of albumen of expressing is larger than the VP5 molecular weight in the virus, be because in the carrier that we add with purification tag (6 * His).
Consisting of of the ELISA test kit of detection GCRV: the monoclonal antibody of embodiment 1 preparation; The solid phase carrier of the polyclonal antibody of coated anti-GCRV; The sheep anti-mouse antibody of horseradish peroxidase mark (available from sigma company); The substrate reactions liquid of enzyme; Positive control; Negative control; Washings; Reaction terminating liquid.
Wherein, the preparation of the polyclonal antibody of goat-anti GCRV and embedding ELISA batten: with the GCRV of gradient centrifugation purification as antigen, the multi-point injection immune goat, be respectively per 1 all immunity once, immunity is 4 times altogether; Get the polyclonal antibody that the serum purifying is anti-GCRV; Adopt the saturated ammonium sulphate method, precipitate 2 times, saturated ammonium sulphate concentration is respectively 50% and 33%, and final precipitation is dissolved with PBS, the dialysis tubing of packing into, in 4 ℃ of lower 72h that dialyse, during every day change liquid at least 4 times.With coated damping fluid (0.05mol/L carbonate buffer solution, pH value 9.6) purifying sheep polyclonal antibody is diluted to concentration 3 μ g/ml, coated 96 hole ELISA enzyme plates (U.S. Corning company), 100 μ l/ holes, 4 ℃ are spent the night.Wash plate 3 times with PBST after taking out, each 5min dries.With the gelatin sealing of 0.01mol/LPBS dilution 1%, every hole 150 μ l, 37 ℃ of sealing 1h.Wash plate 3 times with PBST after taking out, each 3min dries ,-80 ℃ of preservations after the drying.
The preparation of 10 * PBST: NaCl:80 g, Na
2HPO
412H
2O:29g, KH
2PO
4: 2 g, KCl:2g, tween 20: 5 ml, distilled water add to 1000 ml, 4 ℃ of preservations.
The substrate reactions liquid of enzyme: be 10% vitriol (TMB, sigma dispose with dimethyl formamide): 150 μ l, H
2O
2: 4 μ l, pH 5.0 citric acids-phosphoric acid buffer: 10ml.Matching while using.
Positive control is GCRV solution, and negative control is fish cell suspension or healthy tissues homogenate.
Washings is: such as aforementioned PBST preparation.
Reaction terminating liquid: prepare 2mol/LH with distilled water
2SO
4
Embodiment 4: the sandwich ELISA detection method that detects GCRV
The sandwich ELISA method step:
1. (viral titre is 10 to add respectively virus with the amplification of two kinds of methods dilutions to the ELISA enzyme plate of the IgG that is coated with goat-anti GCRV
6TCID50), a kind of method is that the PBS that uses 0.01mol/l dilutes with 10 multiple proportions; Another kind method is to use normal Cyprinus Carpio to dilute.
2. with second antibody, i.e. the monoclonal antibody of the anti-GCRV of embodiment 1 preparation is diluted 1600 times, joins on the enzyme plate.
3. the sheep anti-mouse antibody with the horseradish peroxidase mark dilutes by description of commodity, then joins on the enzyme plate.
4. detersive enzyme target
5. the substrate reactions liquid that adds enzyme
6. termination reaction
The result is as shown in table 2, and the sandwich ELISA method of foundation can be special detects GCRV, and titre can reach 10
5TCID50.
Table 2 detects the detected result of the sandwich ELISA method of GCRV
| P/N | Virus 100 | 10 -1 | 10 -2 | 10 -3 |
| 0.01mol/LPBS virus dilution | 5.45 | 4.32 | 1.73 | 1.24 |
| Normal Cyprinus Carpio virus dilution | 6.79 | 5.08 | 1.73 | 1.24 |
Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here can't give all embodiments exhaustive.Everyly belong to the row that apparent variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Sequence table
＜110〉People's Republic of China Beijing Entry-Exit Inspection and Quarantine Bureau
China Inst. of Quarantine Inspection Sciences
＜120〉monoclonal antibody and the application thereof of anti-GCRV VP5 albumen
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
＜213〉synthetic primer GV-U
<400> 1
gagctcggac ttcgcactct ctctacaatg 30
<210> 2
<211> 27
<212> DNA
＜213〉synthetic primer GV-D
<400> 2
ctcgagaagt tcacgcgggc atggaag 27
Claims (1)
1. secretion produces the hybridoma cell strain of the monoclonal antibody of anti-GCRV VP5 albumen, and its deposit number is CGMCC No.5796.
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| CN104830808A (en) * | 2015-04-27 | 2015-08-12 | 上海海洋大学 | Grass carp reovirus (GCRV) S7 gene mutant strain and applications thereof |
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| CN1616094A (en) * | 2004-09-21 | 2005-05-18 | 中国科学院武汉病毒研究所 | Process for preparing subunit vaccine of reovirus antigen for grass carp |
| CN101864447A (en) * | 2010-05-10 | 2010-10-20 | 中国水产科学研究院长江水产研究所 | Construction of prokaryotic expression vector of grass carp reovirus outer capsid protein and preparation method of polyclonal antibody |
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| CN1616094A (en) * | 2004-09-21 | 2005-05-18 | 中国科学院武汉病毒研究所 | Process for preparing subunit vaccine of reovirus antigen for grass carp |
| CN101864447A (en) * | 2010-05-10 | 2010-10-20 | 中国水产科学研究院长江水产研究所 | Construction of prokaryotic expression vector of grass carp reovirus outer capsid protein and preparation method of polyclonal antibody |
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