CN102266555B - Method for preparing grass carp reovirus (GCRV) gene engineering vaccines - Google Patents
Method for preparing grass carp reovirus (GCRV) gene engineering vaccines Download PDFInfo
- Publication number
- CN102266555B CN102266555B CN201110189906.9A CN201110189906A CN102266555B CN 102266555 B CN102266555 B CN 102266555B CN 201110189906 A CN201110189906 A CN 201110189906A CN 102266555 B CN102266555 B CN 102266555B
- Authority
- CN
- China
- Prior art keywords
- cell
- gcrv
- recombinant virus
- recombinant
- pbs buffer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides a method for preparing grass carp reovirus (GCRV) gene engineering vaccines. The method comprises the following steps of: selecting recombinant baculoviruses (vAcGCRV-VP5/VP7) of sf9 insect cell proliferated recombinant GCRV outer capsid proteins VP5 and VP7; separating recombinant protein components by freezing, thawing and cracking recombinant virus infected sf9 cells and adopting sucrose lining centrifuging method, and thus obtaining a purified in vitro expression VP5 and VP7 protein complex; and performing fish protection experiments on the purified VP5 and VP7 proteins to prove that the VP5 and VP7 proteins have good immune protection effect. The GCRV gene engineering vaccines prepared by the method not only have good stability and immunogenicity, but also have good immune protection effect on grass carp fries; the immune protection rate of the GCRV gene engineering vaccines reaches 90 percent; and the method is suitable for large-scale production and application of the vaccines.
Description
Technical field
The present invention relates to genetic engineering/Molecular Virology field, a kind of GCRV (Grass carp reovirus particularly, abbreviation GCRV, claim again GCHV Grass carp hemorrhagic virus, abbreviation GCHV) the recombinant vaccine preparation method of outer capsid albumen.The present invention's Strain used is GCRV Hunan Shaoyang strain GCRV873, claim again GCHV Hunan Shaoyang strain GCHV873, by the center preservation of Chinese Typical Representative culture collection, preservation date is on July 23rd, 2004, and deposit number is CCTCC NO:V200414.
Background technology
Ctenopharyngodon idellus (Ctenopharyngodon idellus) is one of China fresh water aquaculture thing four large Chinese carps, and it is good and cheap, is deeply subject to liking of consumers in general.GCRV is the main pathogen that causes Ctenopharyngodon idellus death, falls ill when serious, can cause more than 85% Ctenopharyngodon idellus fry Mortality, causes serious economic loss thus to freshwater aquaculture industry.For the control of viral disease, immune protection is effective measures the most, and safe and effective free from environmental pollution.Therefore, for the control of viral disease, adopt vaccine prevention be the most fast, effectively, behave easily.The rough tissue milk of GCRV and cell inactivated vaccine sequential use, in production, and have been obtained certain effect.Although previously also carried out the development of GCRV Antigen Subunit vaccine, Antigen Subunit preparation cost is higher, and have many unfavorable factors at aspects such as the stability of vaccine and safeties, its application is subject to certain limitation.Hemorrhagic disease of grass carp still happens occasionally in some area of China, and the hidden danger that ， China large area is broken out is once again still very important.
In view of rough tissue milk, the hereditary materials such as cell inactivated vaccine and the existing unstability of Antigen Subunit vaccine and remaining nucleic acid, and the virion subunit vaccine cost obtaining with conventional viral purification methods high with the factor such as unstable of tiring, be necessary former preparation method to improve.Confirmed that GCRV outer capsid albumen VP5 and VP7 have good antigenicity, its corresponding antibodies has the viral outer capsid albumen of neutralization, stops the ability of cell entry cell, but the VP5 obtaining by purified virus and VP7 antigen protein are very limited, use genetic engineering means to carry out in vitro this virus protein high efficiency recombinant expressed, preparation GCRV recombinant vaccine can solve the antigen protein limited difficulty of originating.
In recent years, on the basis aspect the researchs such as parsing GCRV873 genome sequence and three dimensional structure and protein characteristic, carry out the preparation of GCRV recombinant vaccine and become possibility.
Summary of the invention
Technical problem to be solved by this invention is: for overcoming above-mentioned the deficiencies in the prior art, a kind of new method is provided, the method uses genetic engineering means to realize in vitro high efficiency recombinant expressed of GCRV albumen, and then prepare reovirus genes of grass carps engineered vaccine, thereby produce and open up new effective way with viral vaccine and commercialization for developing Ctenopharyngodon idellus.
The present invention solves its technical problem and adopts following technical scheme:
Reovirus genes of grass carps engineered vaccine preparation method provided by the invention, its step comprises:
(1) adopt the GCRV outer capsid albumen VP5 of sf9 insect cell line propagation restructuring and the recombinant baculovirus vAcGCRV-VP5/VP7 of VP7;
(2) collect the cell of recombinate shape virus infection, adopt centrifugal, remove supernatant, obtain infecting the sf9 cell of recombinant virus;
(3) cell pyrolysis liquid that adds the PBS buffer by surfactant Triton-X-100 and protease inhibitor PMSF to form in centrifugation, through ice bath and freeze thawing, obtains the recombinant virus cell suspension of cracking.PBS is the english abbreviation of phosphate buffer;
(4) adopt sucrose lining centrifuging, recombinant virus cell suspension is carried out to purification;
(5) with PBS buffer dilution purification VP5 and VP7 albumen composition, make reovirus genes of grass carps engineered vaccine preparation.
The present invention can adopt the method for comprising the following steps to carry out cracking to infecting the sf9 cell of recombinant virus:
(1) collect the sf9 cell of recombinant virus infection 72h, low-speed centrifugal sedimentation cell, to remove culture fluid supernatant; With PBS buffer, rinse cell 3 times, remove bovine serum albumin component;
(2) in the cell precipitation of low-speed centrifugal, add the cell pyrolysis liquid of 3 times of volumes, after fully mixing, ice bath 30min;
(3) freeze thawing treatment cell, puts the freezing cell of quick dissolving in ultralow temperature-80 ℃ refrigerator freezing and 37 ℃ of water-baths;
(4) repeat 2-3 freeze thawing step, obtain the recombinant virus cell suspension of cracking.
Described PBS buffer can be by 137mM NaCl, 2.7mM KCl, 8.1mM Na
2hPO
4with 1.5mM KH
2pO
4form pH7.5.
The PBS buffer that described cell pyrolysis liquid can be 0.5%Triton-X-100 and 1mMPMSF by volumetric concentration forms.
In low-speed centrifugal sedimentation cell process, can employing speed be 8000r/min, centrifugal 20 minutes sedimentation cells.
The present invention can adopt the method for comprising the following steps to carry out purification to the cell suspension of cracking:
(1) cell pyrolysis liquid is added in the cell of centrifugal precipitation, by this mixed liquor ice bath, after 30 minutes, extremely-80 ℃ of ultra cold storage freezers are freezing to put-60 ℃; By the recombinant virus cell suspension of freeze thawing 3 times, with 8000r/min low-speed centrifugal 30 minutes, remove free cell fragment;
(2) suspension of removing cell debris is joined and account in the centrifuge tube of 1/3 volume containing 30% sucrose lining solution, then with 28000r/min ultracentrifugation 2h, the VP5 that centrifugal sediment is vivoexpression and VP7 albumen composition;
(3) the vivoexpression VP5 of purification and VP7 albumen composition are dissolved in PBS buffer by 1:10 times of volume, in-20 ℃, save backup.
The present invention can adopt ultraviolet spectrophotometer to measure vivoexpression VP5 after purification and the content of VP7 albumen composition, and with the dilution of PBS buffer quantitatively, making final concentration is the reovirus genes of grass carps engineered vaccine preparation of 1mg/ml.
The present invention compared with prior art, has following major advantage:
One. expression is high, is suitable for scale and produces in enormous quantities and apply:
Adopt baculovirus expression system, by genetic engineering means, not only obtain GCRV outer capsid albumen VP5 and the high efficiency stable expression of VP7 in Sf9 cell, and vitro recombination protein expression output is high, be therefore suitable for scale and produce in enormous quantities and apply.
They are two years old. there is good stability and immunogenicity:
By sucrose, serve as a contrast centrifugally, purification obtains VP5 and VP7 albumen, has improved the stability of its albumen.The protein product of expressing is non-fusion rotein, therefore has good immunogenicity.
They are three years old. and the sub-vaccine safety of this genetic engineering is good, virus-free nucleic acid compositions.
They are four years old. by immersion treatment fry, can further improve fish body immune protective rate.
In a word, GCRV recombinant vaccine prepared by the present invention not only has good stability and immunogenicity, and Ctenopharyngodon idellus fry is had to good immanoprotection action, and its immune protective rate reaches 90%, is suitable for vaccine scale and produces in enormous quantities and apply.
The specific embodiment
Reovirus genes of grass carps engineered vaccine provided by the invention is to adopt high efficient expression GCRVVP5 in insect cell that gene engineering method obtains and the recombinant vaccine of VP7.The concrete preparation process of its GCRV recombinant vaccine is: by sucrose, serve as a contrast centrifugally, obtain VP5 and the VP7 albumen composition of purification, this purifying protein, with soaking fry after 0.65% normal saline dilution, is obtained to the immune protective effect that reaches 90%.
1. adopt the GCRV outer capsid albumen VP5 of sf9 insect cell line propagation restructuring and the recombinant baculovirus of VP7, collect the sf9 cell of recombinant virus infection 72h, low-speed centrifugal sedimentation cell, removes supernatant.With PBS, be that phosphate buffer rinses 3 times, remove bovine serum albumin component, in the cell precipitation of low-speed centrifugal, add the lysate of 3 times of volumes, after fully mixing, put ultra cold storage freezer freezing.
2. adopt cell pyrolysis liquid (containing the PBS of 0.5%Triton-X100 and 1mM PMSF) and through 3 freeze thawing treatment, the sf9 cell of cracking recombinant virus infection.
3. adopt sucrose lining centrifuging to carry out purification to the virocyte suspension of cracking.
4. the VP5 after purification and VP7 protein sample are measured its content through ultraviolet spectrophotometer, with PBS dilution, are made into the recombinant vaccine preparation that final concentration is 1mg/ml.
Below in conjunction with embodiment, the invention will be further described, but do not limit the present invention.
Reovirus genes of grass carps engineered vaccine preparation of the present invention can adopt the method comprising the following steps:
One. the propagation of recombinant virus and cultivation
Adopt insect Sf 9 cells recombinate transfection and the amplification culture of GCRV capsid protein baculovirus.
The cultivation of 1.Sf9 cell:
Sf9 cell culture culture fluid used is Sf-900SFM serum-free medium (Invitrogen).Get the Sf cell of preserving in liquid nitrogen, put incubation in 37 ℃ of water-baths until ice dissolves.Then on desk centrifuge, after centrifugal 1min, add appropriate Sf-900SFM serum-free medium (Invitrogen), be transferred in culture bottle and cultivate.The suitableeest cultivation temperature of Sf9 cell is 28 ℃, cell culture 2～3 days, until cell degree of converging, reaches after 90-95%, adopts suction pipe piping and druming attached cell to suspended state, is divided into 3 bottles, the cultivation of going down to posterity.
2. the propagation of recombinant virus:
The recombinant virus vAcGCRV-VP5/VP7 cell suspension of preserving going down to posterity is with 10PFU/ml MOI (Plaque-forming unit, PFU: Chinese is plaque forming unit; Multiplicity of infection, MOI: Chinese is infection multiplicity) virus infected cell, infects 72h and collects virocyte suspension.
Two. lysis and expression product purification and evaluation
By the recombinant virus cell suspension of collecting, at 4 ℃ Celsius, with 20 minutes sedimentation cells of 8000r/min low-speed centrifugal.Remove after culture fluid supernatant, with appropriate PBS(137mM NaCl, 2.7mM KCl, 8.1mM Na
2hPO
4, 1.5mMKH
2pO
4; PH7.5) buffer rinses cell 3 times, removes bovine serum albumin component.Then the cell pyrolysis liquid (containing 0.5%Triton X-100(Fluka company product) (Triton X-100) that adds 3 times of volumes in the cell of centrifugation, and 1mMPMSF(Sigma company product) (Phenylmethanesulfonyl fluoride, Chinese: Phenylmethanesulfonyl fluoride)) PBS, after fully mixing, ice bath reaction 30 minutes, put-80 ℃ of refrigerator freezings, then in 37 ℃ of water-baths, dissolve fast freezing cell.By the recombinant virus cell suspension of freeze thawing 3 times, with 8000r/min low-speed centrifugal 30 minutes, remove free cell fragment.The viral suspension of removing cell debris is joined and accounted in the centrifuge tube of 1/3 volume containing 30% sucrose lining solution, with 28000r/min ultracentrifugation 2h, the GCRV VP5 that centrifugation is vivoexpression and VP7 albumen composition.Adopt SDS-PAGE to carry out the evaluation of expression product.
Three. vivoexpression VP5 and VP7 immunogenicity and specificity analyses
Restructuring vivoexpression VP5 after purification and VP7 albumen composition antigen are measured through ultraviolet spectrophotometer (BioteK company product), adopt PBS to be made into final concentration 1mg/ml preparation.Get VP5 and VP7 albumen composition 450 μ g(protein content approximately 1 μ g/ μ l) immunizing rabbit, booster immunization is 2 times after 2 weeks, and dosage is with identical for the first time, every minor tick 14 days.After two weeks, by carotid artery blood-letting, ELISA method detects antibody titer.After determining antibody titer, utilize VP5 and the VP7 prokaryotic expression protein of having prepared and adopt western blotting method to identify its antibody of preparing and VP5 and VP7 albumen to there is good specificity.
Four. neutralization experiment with in and titration
Adopt fixed virus amount--doubling dilution serum method carry out virus in and titration.Experiment arranges 3 groups of repetitions, in 24 hole Corning plates, carries out.Establish viral infection simultaneously and contrast each group with normal cell.Concrete grammar is: with PBS buffer, test serum is made to a series of doubling dilutions, each dilution serum and the virus of having measured are dripped the strain suspension of valency (dripping a valency is 100TCID
50/ 0.1ml) carry out mixed in equal amounts.After every pipe virus antigen and antibody to be measured fully mix, place under 28 ℃ of conditions and hatch 60min.The virus sample that every pipe is got after 0.2ml neutralization is inoculated in the CIK cell being incubated in advance in 24 orifice plates, after absorption 30min, adds certain volume cell maintenance medium (MEM-2), cultivates in 28 ℃, observes day by day, records cytopathy result.The cell that does not add sero-fast virus infected cell and normal uninfecting virus is respectively the positive and negative control.Sero-fast neutralization to be measured is tired and is calculated by Reed-Muech method.Experiment records this antibody neutralization and tires as 1:1280.
The immune protective effect assessment of five .GCRV recombinant vaccines
The vivoexpression VP5/VP7 albumen composition recombinant vaccine of purification is carried out to a series of 10 times of dilutions with 0.65% normal saline, adopt infusion method to soak 30 minutes 3～4cm hatching, obtain good immune protective effect.10
-4dilution VP5/VP7 albumen composition preparation protective rate can reach 90%, 10
-5dilution protective rate is more than 80%.Showing to adopt dilution factor is 10
-4or 10
-5genetic engineering VP5/VP7 albumen composition preparation carries out, after immersion treatment, can reaching the effect of effectively preventing and treating hemorrhagic disease of grass carp to Ctenopharyngodon idellus fry.
In above-described embodiment, adopt the GCRV outer capsid albumen VP5 of sf9 insect cell line propagation restructuring and the recombinant baculovirus (vAcGCRV-VP5/VP7) of VP7, its construction method is mainly: by RT-PCR, increase, obtain respectively the specific fragment of 2 coding GCRV outer capsid albumen VP5 and VP7 genes; By gene clone, screen and obtain the restructuring pFastBacdual dual-expression vector pFbDGCRV-VP5/VP7 that has inserted GCRV VP5 and VP7 gene; The restructuring dual-expression vector that screening is obtained transforms DH10Bac cell, obtains the AcGCRV-VP5/VP7Bacmid of swivel base; And by its transfection Sf 9 insect cell, in Sf9 insect cell, obtain recombinant virus vAcGCRV-VP5/VP7.
Described vAcGCRV-VP5/VP7, its concrete construction method is referring to " expressing recombinant baculovirus and the structure thereof of the GCRV outer capsid albumen " patent documentation (patent No.: 200710168642.2).
Claims (4)
1. a reovirus genes of grass carps engineered vaccine preparation method, adopts the GCRV outer capsid albumen VP5 of sf9 insect cell line propagation restructuring and the recombinant baculovirus vAcGCRV-VP5/VP7 of VP7, it is characterized in that the method comprises the following steps:
(1) collect the cell of recombinate shape virus infection, adopt centrifugal, remove supernatant, obtain infecting the sf9 cell of recombinant virus;
(2) cell pyrolysis liquid that adds the PBS buffer by surfactant Triton-X-100 and protease inhibitor PMSF to form in centrifugation, through ice bath and freeze thawing, obtains the recombinant virus cell suspension of cracking, and concrete grammar is:
To infecting the cleavage method of the sf9 cell of recombinant virus, comprise the following steps:
1) collect the sf9 cell of recombinant virus infection 72h, low-speed centrifugal sedimentation cell, to remove culture fluid supernatant; With PBS buffer, rinse cell 3 times, remove bovine serum albumin component;
2) in the cell precipitation of low-speed centrifugal, add the cell pyrolysis liquid of 3 times of volumes, after fully mixing, ice bath 30min;
3) freeze thawing treatment cell, puts the freezing cell of quick dissolving in ultralow temperature-80 ℃ refrigerator freezing and 37 ℃ of water-baths;
4) repeat 2-3 freeze thawing step, obtain the recombinant virus cell suspension of cracking;
(3) adopt sucrose lining centrifuging, recombinant virus cell suspension is carried out to purification, its step comprises:
1) cell pyrolysis liquid is added in the cell of centrifugal precipitation, by this mixed liquor ice bath, after 30 minutes, extremely-80 ℃ of ultra cold storage freezers are freezing to put-60 ℃; By the recombinant virus cell suspension of freeze thawing 3 times, with 8000r/min low-speed centrifugal 30 minutes, remove free cell fragment,
2) suspension of removing cell debris is joined and account in the centrifuge tube of 1/3 volume containing 30% sucrose lining solution, then with 28000r/min ultracentrifugation 2h, the VP5 that centrifugal sediment is vivoexpression and VP7 albumen composition,
3) the vivoexpression VP5 of purification and VP7 albumen composition are dissolved in PBS buffer by 1:10 times of volume, in-20 ℃, save backup; Described PBS buffer is by 137mM NaCl, 2.7mM KCl, 8.1mM Na
2hPO
4and 1.5mMKH
2pO
4form pH7.5;
(4) with after PBS buffer dilution purification VP5 and VP7 albumen composition, be made into the reovirus genes of grass carps engineered vaccine preparation of final concentration 1mg/ml.
2. reovirus genes of grass carps engineered vaccine preparation method according to claim 1, is characterized in that the PBS buffer that cell pyrolysis liquid is 0.5%Triton-X-100 and 1mMPMSF by volumetric concentration forms.
3. reovirus genes of grass carps engineered vaccine preparation method according to claim 1, is characterized in that employing speed is 8000r/min, centrifugal 20 minutes sedimentation cells.
4. reovirus genes of grass carps engineered vaccine preparation method according to claim 1, it is characterized in that adopting ultraviolet spectrophotometer to measure vivoexpression VP5 after purification and the content of VP7 albumen composition, with the dilution of PBS buffer quantitatively, making final concentration is the reovirus genes of grass carps engineered vaccine preparation of 1mg/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110189906.9A CN102266555B (en) | 2011-07-08 | 2011-07-08 | Method for preparing grass carp reovirus (GCRV) gene engineering vaccines |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110189906.9A CN102266555B (en) | 2011-07-08 | 2011-07-08 | Method for preparing grass carp reovirus (GCRV) gene engineering vaccines |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102266555A CN102266555A (en) | 2011-12-07 |
| CN102266555B true CN102266555B (en) | 2014-03-12 |
Family
ID=45049135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110189906.9A Expired - Fee Related CN102266555B (en) | 2011-07-08 | 2011-07-08 | Method for preparing grass carp reovirus (GCRV) gene engineering vaccines |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102266555B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881155A (en) * | 2017-11-21 | 2018-04-06 | 上海海洋大学 | Express GCRV spike protein VP55 recombinant baculovirus and application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102755652A (en) * | 2012-07-04 | 2012-10-31 | 鲁东大学 | Bivalent DNA (Deoxyribonucleic Acid) vaccine and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101205545A (en) * | 2007-12-06 | 2008-06-25 | 中国科学院武汉病毒研究所 | Recombinant baculoviral for expressing grass carp reovirus external casing protein and construction thereof |
-
2011
- 2011-07-08 CN CN201110189906.9A patent/CN102266555B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101205545A (en) * | 2007-12-06 | 2008-06-25 | 中国科学院武汉病毒研究所 | Recombinant baculoviral for expressing grass carp reovirus external casing protein and construction thereof |
Non-Patent Citations (6)
| Title |
|---|
| Expression of Outer Capsid Protein VP7 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity;Lan-lan ZHANG等;《VIROLOGICA SINICA》;20091231;第24卷(第6期);第545-551页 * |
| High Level Expression of Grass Carp Reovirus VP7 protein in Prokaryotic Cells;Lan-lan ZHANG等;《VIROLOGICA SINICA》;20080229;第23卷(第1期);第51-56页 * |
| Lan-lan ZHANG等.Expression of Outer Capsid Protein VP7 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity.《VIROLOGICA SINICA》.2009,第24卷(第6期),第545-551页. |
| Lan-lan ZHANG等.High Level Expression of Grass Carp Reovirus VP7 protein in Prokaryotic Cells.《VIROLOGICA SINICA》.2008,第23卷(第1期),第51-56页. |
| 方勤等.草鱼呼肠孤病毒的三位重构与衣壳蛋白特性.《中国科学C辑》.2005,第35卷(第3期),第231-237页. |
| 草鱼呼肠孤病毒的三位重构与衣壳蛋白特性;方勤等;《中国科学C辑》;20051231;第35卷(第3期);第231-237页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881155A (en) * | 2017-11-21 | 2018-04-06 | 上海海洋大学 | Express GCRV spike protein VP55 recombinant baculovirus and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102266555A (en) | 2011-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022144040A1 (en) | Nucleotide sequence encoding novel coronavirus antigen, and use thereof | |
| CN103585625B (en) | A kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application | |
| CN103266090B (en) | Asia1 type foot-and-mouth disease recombinant virus and preparation method and application thereof | |
| CN102321639B (en) | Preparation method of virus-like particles (VLPs) of Chikungunya virus (CHIKV) and its application | |
| CN109536461B (en) | O-type foot-and-mouth disease virus mutant strain and preparation method and application thereof | |
| CN101695569A (en) | Univalent and bivalent gene engineered subunit vaccine for hand-foot-and-mouth disease and preparation method thereof | |
| WO2016086576A1 (en) | Vector expressing poliomyelitis virus-like granule protein and method for preparing poliomyelitis virus-like granules | |
| CN102220293B (en) | Dog flu recombinant virus as well as preparation method and application thereof | |
| CN109321535A (en) | A kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain | |
| CN104152416A (en) | Pseudorabies virus gene-deleted attenuated strain as well as preparation method and application thereof | |
| CN104611299B (en) | H9N2 avian flus strain, preparation method, vaccine combination and its application of a kind of artificial recombination | |
| CN102266555B (en) | Method for preparing grass carp reovirus (GCRV) gene engineering vaccines | |
| CN105274142A (en) | Replicative recombinant human 55-type Adenovirus vector and preparation method and application thereof | |
| Yan et al. | Phylogenetic analysis of S1 gene of infectious bronchitis virus isolates from China | |
| CN104419686B (en) | recombinant PRRS virus HV-nsp9 and application thereof | |
| CN106367398B (en) | A kind of 16 type Strain of human coxsackievirus A group and its preparing the application in inactivated vaccine | |
| CN103614345B (en) | A kind of influenza virus vaccine strain | |
| Graham et al. | Cultural characteristics of salmonid alphaviruses–influence of cell line and temperature | |
| CN103014043B (en) | Safe carrier for seed viruses of inactivated vaccine for foot-and-mouth disease and application thereof | |
| CN104436187A (en) | Vaccine for expressing rabbit hemorrhagic disease virus VP60 protein | |
| CN109536464B (en) | Chikungunya virus infectious clone with deletion of capsid protein gene, construction method and application in preparation of attenuated vaccine | |
| JP2022539511A (en) | Virus production method and collection solution composition | |
| CN1306959C (en) | Process for preparing subunit vaccine of reovirus antigen for grass carp | |
| CN102703475B (en) | Toxicity weakened NSP4 mutant gene, recombinant plasmid and recombinant bovine rotavirus | |
| CN102190701B (en) | Method for separating and purifying influenza virus hemagglutinin on large scale |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140312 Termination date: 20200708 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |