CN1736481A - Recombined chicken pox virus vaccine rPFV-12LSH9A, preparation process and use thereof - Google Patents
Recombined chicken pox virus vaccine rPFV-12LSH9A, preparation process and use thereof Download PDFInfo
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Abstract
Disclosed is a recombined chicken pox virus vaccine rPFV-12LSH9A, preparation process and use, wherein the construction process consists of placing AIV HA gene fragment containing H9 hypotype onto the downstream of right promotors, inserting into chicken poxviridae expression vector p12-18 containing FPV duplicated non-indispensable fragment FPV1-2 and reported genes to construct transition vector, then transforming chick embryo desmocyte infected with wt-FPV with the transition vector, and obtaining rFPV expressing H9 hypotype AIV HA gene through consanguinity recombination, i.e. recombinant chicken poxviridae vaccine rFPV-12LSH9A.
Description
Technical field
The present invention relates to a kind of recombinant fowlpox vaccine virus, particularly a kind of with the new recombinant fowlpox vaccine virus of identifying that duplicates nonessential region construction expression H9 subtype avian influenza virus hemagglutinin gene of bird pox virus.
Background technology
(Fowlpox Virus, be called for short: FPV) be the member of Poxviridae, Avipoxvirus, be a kind of virus maximum in the animal virus of finding so far to bird pox virus.Unique character is that they duplicate at the endochylema of infection cell, and not in nucleus.Sophisticated virion is the brick type, and size is 250 * 350nm, and the genome of FPV is bifilar linear DNA, the about 300kb of size, and molecular weight is about 2～4 * 10
5KD, G+C content reaches 35%, and its DNA does not have infectivity.By recombinant DNA technology, be carrier development birds genetic engineering reorganization live-virus vaccine with FPV, or mammal non-replicating reorganization live vector vaccine is subjected to the attention of more and more researcheres.FPV carrier and the viral vector of having reported such as vaccinia virus, papillomavirus, adenovirus, parvovirus, marek's disease virus, tobacco mosaic virus (TMV) etc. are compared has remarkable advantages.An infected poultry under the FPV natural conditions can produce abortive infection at mammalian cell, though do not produced infective progeny virus particle, but can synthesize the processing exogenous antigen automatically and offer cell surface, stimulates body to produce immune response.
(recombinantfowlpox virus obtains expressing in rFPV) protein of existing polytype biologically active at recombinant Borrel virus.Hemagglutinin gene (HA) as bird flu virus (AIV); the fusion rotein (F) of Avian pneumo-encephalitis virus (NDV) and hemagglutinin-neuraminidase (HN) gene; the gB gene of marek's disease virus (MDV); the VP2 gene of infectious bursal disease virus (IBDV); rabies virus glucoprotein and Measles virus antigen-4 fusion protein gene etc.; rFPV with expression alien gene carries out immunity; all can provide specific immunoprotection SPF chicken or the commercial chicken that do not have a maternal antibody; therefore; the FPV carrier not only can be used as the vaccine of poultry, and can be used as mammiferous vaccine.
In the prior art, the FPV vaccine used in commodity fowl more than 70 year, slight, self limiting local infection that this vaccine can cause.Simultaneously, the FPV host range is narrower, only infects fowl, is relatively safe vaccine virus therefore.
Fowlpox virus vector after deliberation recent two decades, but it is very few to be applied to clinical recombinant virus why? a main cause is exactly the interference of maternal antibody.The maternal antibody of commercial chicken is also very high, wherein at the maternal antibody of H9N2 hypotype AIV up to 2
7-2
8, the maternal antibody of H5N1 hypotype AIV has also reached 2
5-2
6, this makes troubles just for the use of commercial chicken vaccine and the monitoring of epidemic situation.
Discoveries such as Taylor, on the test chicken that higher ND maternal antibody is arranged, the immune protective rate of the opposing NDV strong virus attack of the reorganization FPV of coexpression NDVF and HN gene descends on the SPF chicken to some extent.Therefore, at the antibody interferes with of exogenous gene the immunity of reorganization FPV.No matter whether 9 reorganization FPV that express the HPAIV gene that this chamber makes up altogether can induce HI antibody; the attack that on the SPF chicken, all can resist strong malicious AIV; immune protective rate is 100%; 3 the coexpression HPAIV that make up and the reorganization Seedling of MPAIVHA gene all can suppress the discharge of MPAIV on the SPF chicken, immune efficacy and oil seepage are suitable.And on the commercial chicken that has high-level AIV maternal antibody, protective rate all has decline in various degree.Show at the maternal antibody of foreign protein there is certain influence really in the immune efficacy of reorganization FPV.The commercial chicken test result of the test that other has the recombinant viral vaccine of main protection antigen of the NDV of structure to carry out also shows; the immune efficacy of reorganization FPV is subjected to very big influence (Ding Weidong because of there being the maternal antibody at NDV or AIV; Cao Liping; Zhang Tiyin etc. express the hereditary stability and the immuning effect test of Avian pneumo-encephalitis virus F and HN gene recombinaton bird pox virus vaccine. Chinese veterinary's magazine; 2005; 41:10-14. Wei Dong is flat; Liu Yuliang; the Shao Wei magnitude. the recombinant Borrel virus of coexpression newcastle F gene and H9 subtype avian influenza virus HA gene. the microorganism journal; 2004,44:14-18.).
In order to obtain clone and the screening that ideal recombinant virus at first must carry out the virus replication nonessential region, exogenous gene only is inserted into the expression that nonessential region just might be realized protein product of duplicating of viral gene, and the vector expression exogenous gene directly is subjected to its restriction of inserting the site in carrier, so the acquisition of nonessential region is the prerequisite of construction of recombinant virus.The virus replication nonessential region has the strict nonessential region and the branch of general nonessential region again, must select the strict nonessential region that duplicates when construction of recombinant virus, just can keep the replication capacity of parent plant and the immune efficacy of assurance recombinant virus.
Discover that the nonessential region FPV7S that duplicates of the insertion carrier p11S that this chamber made up in the past just in time is on the open reading frame, may destroy the IL-18 binding-protein gene.At present existing scholar finds, through the conjugated protein generation that can suppress body IL-18 dependency IFN-γ of the IL-18 of cellular expression.Checking is real factually, IL-18 is the inflammatory cytokine that causes that has multiple function in the IL-1 family, it can be induced body to produce IFN-γ, Th-1 type immunne response and activate the NK cell activity, and this induces the immunne response that produces effective anti-VV infection to play important effect to mice.And the conjugated protein function that may have the reaction of antagonism body inflammatory of IL-18 is favourable in the intravital breeding of host with diffusion to virus.
In order to solve this defective, Sun Lei, Liu Wujie, what Chen Sujuan etc. had screened the bird pox virus strictness again duplicates nonessential region FPV1-2, made up bird pox virus expression vector p12-18, carrier is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 6th, 2005, and preserving number is: CGMCC NO:1385, its length is: 10450bp.
Summary of the invention
First purpose of the present invention is in order to overcome the deficiency of existing carrier, can increase the practicality that the recombinant Borrel virus recombinant vaccine uses the commercial chicken group, keep simultaneously and strengthen the immunogenicity that the immunogenicity of recombinant Borrel virus and external source are inserted the expression of gene product, can use new bird pox virus expression vector expression alien gene, make recombinant fowl pox virus live vaccine commercialization early, a kind of recombinant Borrel virus recombinant vaccine that can use the male commercial chicken group of maternal antibody is provided.
Vaccine of the present invention is to contain the recombinant fowlpox vaccine virus of expressing H9 hypotype AIV HA gene, and on June 17th, 2005 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is: CGMCC 1390.
Exogenous gene in the recombinant Borrel virus of the present invention is the hemagglutinin gene of H9 subtype avian influenza virus, not disturbed by maternal antibody, can increase the practicality that the recombinant Borrel virus recombinant vaccine uses the commercial chicken group, keep simultaneously and strengthen the immunogenicity that the immunogenicity of recombinant Borrel virus and external source are inserted the expression of gene product, it is clinical to make that recombinant fowl pox virus live vaccine is applied to early.
Second purpose of the present invention provides the construction method of a kind of recombinant fowlpox vaccine virus rFPV-12LSH9A:
May further comprise the steps:
(1) acquisition contains the genetic fragment of the whole reading frame of H9 hypotype AIV HA gene.
(2) the said gene fragment is placed suitable promoter downstream, be inserted among the bird pox virus expression vector p12-18 that contains FPV replicate nonessential fragment FPV1-2 and reporter gene (LacZ) and be built into transfer vector;
(3) infected the chick embryo fibroblast (CEF) of wt-FPV with the transfer vector transfection, obtained to express the rFPV of H9 hypotype AIV HA gene, i.e. recombinant fowlpox vaccine virus rFPV-12LSH9A by homologous recombination.
Can be subjected to the interferential recombinant fowl pox virus live vaccine of maternal antibody by above method, its methodological science, good reliability, the vaccine stability of acquisition is good, the purity height.
H9 hypotype AIV HA gene can obtain by the following method in the above-mentioned steps (1): with 9-10 age in days SPF Embryo Gallus domesticus amplicon virus, collect allantoic fluid, phenol-virus genomic the RNA of SDS method extracting, design a pair of primer according to the H9 hypotype AIV HA gene order of having delivered:
PH9A1 5’-GCTGGATCCGCCACCATGGAAACAATATCACTAATAGCT-3’
PH9A2 5’-GTCGGATCCAAGCTTACAAAAAGCCAATTATATACAAATCTTGC-3’
The synthetic cDNA of elder generation's reverse transcription, pcr amplification goes out the whole reading frame of H9 hypotype AIV HA gene then.H9 hypotype AIV HA gene can be connected to the T-carrier, the correctness of the gene order of order-checking proof gained.
Promoter can be any FPV promoter, vaccinia virus promoter or synthetic promoter that can instruct genetic transcription in recombinant Borrel virus (rFPV) infection cell in the step (2).
In the step (2) promoter and said gene by the enzyme action adjunction method or link together with the method for PCR design primer adjunction, be inserted into according to a conventional method and contain the FPV that FPV duplicates nonessential fragment FPV1-2 and reporter gene LacZ and insert in the carrier, this connection product is being transformed in the appropriate host cell, purification and analyze recombinant obtains transfer vector p12LSH9A according to a conventional method.Can prepare the plasmid DNA of transfer vector and with the PEG plasmid DNA purification by the alkaline lysis method.
The construction method of transfer vector p12LSH9A is: through klenow enzyme mend flat with the HindIII enzyme action plasmid pTH9A, phenol: the line style plasmid DNA is reclaimed in the chloroform extracting, reuse BamHI restriction enzyme digestion and electrophoresis reclaims the 1.7kb fragment, is connected with vector plasmid p12-18 with SmaI and BamHI enzyme action respectively.
Step is used GeneJammer in (3)
TMTransfection reagent with transfer vector respectively with wt-FPV cotransfection chick embryo fibroblast (CEF), the CEF that the inoculation of supernatant diluent is grown up to fine and close monolayer, after waiting to occur single typical plaque, cover with the Nutrient agar that contains X-gal, locus coeruleus screening purification gets recombinant virus rFPV-12LSH9A.The insertion of exogenous gene and expression among the rFPV can be identified by the methods such as biological characteristics of nucleic acid hybridization, pcr amplification exogenous gene, recombinant virus and bonded reactivity of specific antibody and expressed exogenous genes products.
The rFPV that the present invention also will express H9 hypotype AIV HA gene breeds on the CEF monolayer, makes virus amplification in a large number in CEF.Gather in the crops after growing up to complete pathological changes, draw 0.1ml virus liquid respectively, do continuous 10 times of dilutions, get 10 with cell culture fluid
-4, 10
-5, 10
-6Three dilution viral liquid inoculations grow in the CEF of the fine and close monolayer of formation in the 24 porocyte culture plates, and each dilution factor is inoculated 4 holes, every hole inoculation 0.1ml virus liquid.Cultivate 72h for 37 ℃, observe pathological changes then, the CEF that calculates each dilution factor virus liquid of inoculation goes up the plaque number that forms, and calculates the viral plaque forming unit (PFU) that contains in the virus stock solution used according to count results.Inoculation experiments is used animal.
The 3rd purpose of the present invention provides the purposes of recombinant fowlpox vaccine virus rFPV-12LSH9A: be used to prevent and treat the H9 subtype avian influenza virus.Corium Gallus domesticus is injection down, and injection volume is 10
4-10
6PFU/ only.
This method clinical manipulation, side effect is little, does not influence the epidemiology epidemic monitoring.
Description of drawings
Fig. 1 is the pcr amplification product electrophoresis pattern of H9 hypotype AIV HA gene.
Among the figure, Fig1:H9A gene amplified by PCR;
M：200bp?DNA?ladder?maker；
1:F strain H9A gene PCR product.
Fig. 2 is that the enzyme action of recombiant plasmid p12LSH9A is identified electrophoresis pattern.
Among the figure, M:DNA Maker III;
1:EcoRI digests p12LSH9A.
Fig. 3 is for making up the sketch map of the transfer vector p12LSH9A that contains H9 hypotype AIV HA gene.
The blue color plaque photo of Fig. 4 for forming behind the purification of Recombinant viral infection CEF.
Fig. 5 is the fluorogram of expressing exogenous proteins behind the recombinant virus infection CEF.
Fig. 6 is the sequence of H9 hypotype AIV HA gene.
The specific embodiment
Specify below with reference to examples of implementation and accompanying drawing, but routine examples of implementation are further to explain rather than restriction the present invention down.
Relate to bird pox virus expression vector p12-18 in the present embodiment, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 6th, 2005, preserving number is: CGMCC NO:1385, its length is: 10450bp.
Step 1: amplification, clone and the sequence analysis of H9 hypotype AIV HA gene
According to the sequence of this laboratory at known H9 hypotype AIV HA, the pcr amplification primer of design H9N2 hypotype AIV HA gene.Primer is synthetic by precious biological engineering (Dalian) company limited.
The amplimer of H9A gene: the 5 ' end of forward primer PH9A1 is introduced BamHI site and Kozak sequence (shown in square frame); 5 ' the end of downstream primer PH9A2 is introduced outside BamHI, the HindIII site, has also introduced the transcription stop signals (shown in square frame) of poxvirus genome group early gene.The upstream and downstream primer is striden the width of cloth and is about 1.7kb, covers whole H9A gene code frame.
PH9A1 5’-GCTGGATCCGCCACCATGGAAACAATATCACTAATAGCT-3’
BamHI
PH9A2 5’-GTCGGATCCAAGCTTACAAAAAGCCAATTATATACAAATCTTGC-3’
BamHI?HindIII
With plasmid pTH9A is template, with PH9A1 and PH9A2 primer and Expand High FidelityPCR system the H9A gene is carried out pcr amplification, connect one section Kozak sequence before making H9A gene translation sintering, connect poxvirus early transcription termination signal T5NT after the termination codon.PCR loop parameter: 94 ℃ of pre-degeneration 5min; 94 ℃ of degeneration 1min, 58 ℃ of annealing 1min, 72 ℃ are extended 2min, carry out 30 circulations altogether; 72 ℃ are extended 10min again after the loop ends.The PCR product reclaims and purification, and is connected with pGEM-T easy vector after identifying by electrophoresis.With the screening of BamHI+HindIII double digestion method, with the positive recombiant plasmid of the plasmid that cuts out the 1.7Kb size strip, called after pTH9A.Positive recombiant plasmid send precious biological engineering (Dalian) the company limited conclusive evidence that checks order.
Step 2: the structure of transfer vector (p12LSH9A)
Construction strategy such as Fig. 3 of transfer vector (p12LSH9A).
Through klenow enzyme mend flat with the HindIII enzyme action plasmid pTH9A, phenol: the line style plasmid DNA is reclaimed in the chloroform extracting, reuse BamHI restriction enzyme digestion and electrophoresis reclaims the 1.7kb fragment, is connected with bird pox virus expression vector p12-18 with SmaI and BamHI enzyme action respectively, is built into transfer vector p12LSH9A.
Step 3: transfection and purification
GeneJammer is pressed in transfection
TMThe explanation of 6 transfection reagents is carried out.Cultivate the SPF fibroblast to forming monolayer in the 60mm culture dish, infect CEF with the wt-FPV vaccine strain of 0.1MOI, cultivate 3～4h for 37 ℃, it is standby to add 4ml after going supernatant with the basic basic washed twice of DMEM.1-2 μ g p12LSH9A is dissolved in the TE of 30 μ l respectively, by 1: 6 with GeneJammer
TM6 are diluted in the 100 μ l DMEM basal mediums, and mixing is put room temperature effect 15min gently, and the said mixture of p12LSH9A is slowly dropped to mixing in the DMEM basal medium that has infected wt-FPVCEF; Cultivate to change behind the 6h for 37 ℃ and keep liquid, treat results virus after the complete pathological changes of cell ,-70 ℃～37 ℃ multigelations 3 times, low-speed centrifugal is removed cell debris, and supernatant is used for the screening of recombinant virus.
The CEF that the inoculation of supernatant diluent is grown up to fine and close monolayer, after waiting to occur single typical plaque, cover with the Nutrient agar that contains 200 μ g/ml X-gal, cultivate 72h for 37 ℃, wait to occur the further clone purification of the blue plaque of picking behind the blue plaque, under the condition that exists at X-gal, it is blue that recombinant virus formed plaque on CEF all is.
Step 4: express the evaluation of the different recombinant Borrel virus of H9 hypotype AIV HA gene
Infect to have formed the CEF of fine and close monolayer with rFPV-12LSH9A, and establish rFPV-11SH9A respectively and wt-FPV is positive and negative control.At 37 ℃, 5%CO
2Cultivate under the condition after typical plaque occurring, the culture fluid that inclines, with PBS (0.01M, pH7.4) gently washing once, then with the fixing 10min of cold methanol; Adding dilution factor is the monoclonal antibody of 1: 1000 mouse-anti H9 hypotype AIV F strain, hatches 1h for 37 ℃; (be called for short PBST, pH7.2) wash 5min * 3 time, add dilution factor again and be sheep anti-mouse igg-FITC fluorescent antibody of 1: 200, hatch 1h for 37 ℃ with the 0.01M PBS that contains 0.05% tween 80; Wash observation and Taking Pictures recording result under time rearmounted fluorescence microscope of 5min * 3 with PBST.
Step 5: the immune efficacy experiment of recombinant virus
1, the immanoprotection action that opposing H9 hypotype AIV infects behind the SPF experimental chicken inoculation rFPVs (is index with the 5th day toxin expelling behind the counteracting toxic substances)
7 age in days Bai Laihang SPF chickling random packet are respectively at the subcutaneous immune rFPV-11SH9A of cervical region, rFPV-12LSH9A and wt-FPV, 10
4-10
6PFU/ only.PBS matched group and oil-emulsion inactivated vaccine matched group, 0.2ml/ are only.After immunity 7,10,14,18, blood sampling in 21 days, separation of serum detects the HI antibody titer.Found that various recombiant vaccinies all can induce the specificity HI antibody at H9 hypotype AIV.Vaccine by the p12-18 vector construction is slightly higher than the HI antibody horizontal that the corresponding vaccine by the p11S vector construction induces.
In the immunoprotection test of SPF chicken, rFPV
LPThe PI of-11SH9A and rFPV-12LSH9A is respectively 77.78 and 100.
The immanoprotection action that opposing H9 hypotype AIV infects behind the table 1.SPF chicken inoculation rFPVs
| Group | Immunizing dose (PFU/ only) | Counteracting toxic substances dosage (ELD 50/ valency) | The counteracting toxic substances approach | The virus separation rate | Protection index (PI) |
| The newborn inactivated vaccine rFPV-11SH9A rFPV-12LSH9A wt-FPV PBS of H9 hypotype AIV oil | ?10 5?10 5?10 5?10 5?10 5 | 10 7 10 7 10 7 10 7 10 7 | Collunarium eye dripping collunarium eye dripping collunarium eye dripping collunarium eye dripping collunarium eye dripping | 1/10 2/10 0/10 9/10 9/10 | 88.89 77.78 100 - - |
2, rFPVs is at the immune efficacy (the 5th day toxin expelling is an index after with counteracting toxic substances) of commercial chicken
In the immunoprotection test of commercial chicken, the PI of oil-emulsion inactivated vaccine is 83; The PI of rFPV-11SH9A and rFPV-12LSH9A is respectively 75 and 83.By the recombiant vaccine of p12-18 vector construction respectively than the immune efficacy by the recombiant vaccine of p11S vector construction is slightly high accordingly.
The immunoprotection test that opposing H9 hypotype AIV infects behind the table 2. commodity egg inoculation rFPVs
| Group | Immunizing dose (PFU/ only) | Counteracting toxic substances dosage (ELD 50/ only) | The counteracting toxic substances approach | The virus separation rate | Protection index (PI) |
| The newborn inactivated vaccine rFPV-11SH9A rFPV-12LSH9A wt-FPV PBS of H9 hypotype | 10 5 10 5 10 5 10 5 10 5 | ?10 7?10 7?10 7?10 7?10 7 | Collunarium eye dripping collunarium eye dripping collunarium eye dripping collunarium eye dripping collunarium eye dripping | 2/26 3/26 2/26 12/26 12/26 | 83 75 83 - - |
Claims (9)
1, recombinant fowlpox vaccine virus rFPV-12LSH9A is characterized in that this vaccine is to contain the recombinant fowlpox vaccine virus of expressing H9 hypotype AIV HA gene, and preserving number is: CGMCC 1390.
2, the preparation method of recombinant fowlpox vaccine virus rFPV-12LSH9A is characterized in that may further comprise the steps:
1) acquisition contains the genetic fragment of the whole reading frame of H9 hypotype AIV HA gene;
2) the said gene fragment is placed suitable promoter downstream, be inserted among the bird pox virus expression vector p12-18 that contains FPV replicate nonessential fragment FPV1-2 and reporter gene (LacZ) and be built into transfer vector;
3) infected the chick embryo fibroblast (CEF) of wt-FPV with the transfer vector transfection, obtained to express the rFPV of H9 hypotype AIV HA gene, i.e. recombinant fowlpox vaccine virus rFPV-12LSH9A by homologous recombination.
3, according to the preparation method of the described recombinant fowlpox vaccine virus rFPV-12LSH9A of claim 2, it is characterized in that H9 hypotype AIV HA gene can obtain by the following method in the step 1): with 9-10 age in days SPF Embryo Gallus domesticus amplicon virus, collect allantoic fluid, phenol-virus genomic the RNA of SDS method extracting is by following primer
PH9A1 5’-GCTGGATCCGCCACCATGGAAACAATATCACTAATAGCT-3’
PH9A2 5’-GTCGGATCCAAGCTTACAAAAAGCCAATTATATACAAATCTTGC-3’
The synthetic cDNA of elder generation's reverse transcription, pcr amplification goes out the whole reading frame of H9 hypotype AIV HA gene then.
4, according to the preparation method of the described recombinant fowlpox vaccine virus rFPV-12LSH9A of claim 2, it is characterized in that step 2) in promoter can be any FPV promoter, vaccinia virus promoter or synthetic promoter that can in recombinant Borrel virus (rFPV) infection cell, instruct genetic transcription.
5, according to the preparation method of claim 2 or 4 described recombinant fowlpox vaccine virus rFPV-12LSH9A, it is characterized in that step 2) in promoter and said gene by the enzyme action adjunction method or link together with the method for PCR design primer adjunction, be inserted into according to a conventional method and contain the FPV that FPV duplicates nonessential fragment FPV1-2 and reporter gene LacZ and insert in the carrier, this connection product is being transformed in the appropriate host cell, purification and analyze recombinant obtains transfer vector p12LSH9A according to a conventional method.
6, according to the preparation method of the described recombinant fowlpox vaccine virus rFPV-12LSH9A of claim 5, the construction method that it is characterized in that transfer vector p12LSH9A is: through klenow enzyme mend flat with the HindIII enzyme action plasmid pTH9A, phenol: the line style plasmid DNA is reclaimed in the chloroform extracting, reuse BamHI restriction enzyme digestion and electrophoresis reclaims the 1.7kb fragment, is connected with vector plasmid p12-18 with SmaI and BamHI enzyme action respectively.
7,, it is characterized in that using in the step 3) GeneJammer according to the preparation method of the described recombinant fowlpox vaccine virus rFPV-12LSH9A of claim 2
TMTransfection reagent with transfer vector respectively with wt-FPV cotransfection chick embryo fibroblast (CEF), the CEF that the inoculation of supernatant diluent is grown up to fine and close monolayer, after waiting to occur single typical plaque, cover with the Nutrient agar that contains X-gal, locus coeruleus screening purification gets recombinant virus rFPV-12LSH9A.
8,, it is characterized in that the rFPV that will express H9 hypotype AIV HA gene breeds, and makes virus amplification in a large number in CEF on the CEF monolayer according to the preparation method of the described recombinant fowlpox vaccine virus rFPV-12LSH9A of claim 2.
9, the purposes of recombinant fowlpox vaccine virus rFPV-12LSH9A is characterized in that being used to prevent and treat the H9 subtype avian influenza.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140441A (en) * | 2010-11-04 | 2011-08-03 | 高福 | Bird flu vaccine by using attenuated vaccinia Tian Tan as vector |
| CN107475297A (en) * | 2017-06-20 | 2017-12-15 | 广东温氏食品集团股份有限公司 | A kind of recombinant Borrel virus transfer vector for expressing the type adenovirus fiber2 genes of duck 2 and its construction method and application |
-
2005
- 2005-08-08 CN CN 200510041454 patent/CN1736481A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140441A (en) * | 2010-11-04 | 2011-08-03 | 高福 | Bird flu vaccine by using attenuated vaccinia Tian Tan as vector |
| CN107475297A (en) * | 2017-06-20 | 2017-12-15 | 广东温氏食品集团股份有限公司 | A kind of recombinant Borrel virus transfer vector for expressing the type adenovirus fiber2 genes of duck 2 and its construction method and application |
| CN107475297B (en) * | 2017-06-20 | 2020-12-01 | 温氏食品集团股份有限公司 | Recombinant fowlpox virus transfer vector for expressing duck type 2 adenovirus fiber2 gene and construction method and application thereof |
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