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Chicken infectious anemia virus VP1 and VP2 expression genetic recombination fowlpox virus living-vector vaccine

A chicken infectious anemia, live vector vaccine technology, applied in the direction of virus/phage, vaccine, virus, etc., can solve the problems of high cost, low virus titer, rare use of inactivated vaccine, etc., to reduce economic losses, Avoid the effect of horizontal transmission

Active Publication Date: 2019-01-04
广州金苗动保科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, CAV inactivated vaccines have been successfully developed both at home and abroad, and they have certain protective ability to offspring chickens of immunized breeders, but due to the low titer of the virus in chicken embryos and cells (the antigen content in the vaccine is generally low at 10 5 CID 50 / ml), it is necessary to pay a higher cost to obtain sufficient virus titers, so the use of inactivated vaccines in production is rare
However, there are no suitable commercial products for live vaccine strains in China, and genetically engineered vaccines are a promising vaccine direction
Noteborn, Xiao Qingli, etc. co-expressed CAV VP1 and VP2 in insects and successfully induced antibody production, but this achievement is still in the laboratory research stage

Method used

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  • Chicken infectious anemia virus VP1 and VP2 expression genetic recombination fowlpox virus living-vector vaccine
  • Chicken infectious anemia virus VP1 and VP2 expression genetic recombination fowlpox virus living-vector vaccine
  • Chicken infectious anemia virus VP1 and VP2 expression genetic recombination fowlpox virus living-vector vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Chicken Infectious Anemia Virus VP1, VP2 Gene Recombination Fowlpox Virus Live Vector Vaccine rFPV-VP1-VP2 Construction

[0031] a. Isolation and identification of CAV epidemic strains

[0032] The nucleotide sequences of 36 chicken infectious anemia virus reference strains isolated from around the world were downloaded from the NCBI database, and compared with the whole genome sequences of 12 chicken infectious anemia virus strains recently isolated in Guangdong by our laboratory. Using MegAlign software, the homology comparative analysis was carried out on the complete gene sequences, VP1 gene nucleotide sequences and deduced VP1 amino acid sequences of 12 isolates and 36 reference viruses. The results of evolutionary analysis of the whole gene sequence showed that the phylogenetic tree was composed of three branches: branch II included Chinese isolates DQ141673 and AY999018; branch III included Chinese isolates DQ124936 and KU221054, American isolate AF3119...

Embodiment 2

[0049] Example 2: Titer and Growth Curve Determination of Recombinant Fowlpox Virus

[0050] a. Titer of recombinant fowlpox virus

[0051] Take 100 μL and dilute to 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 The virus solution of the recombinant fowlpox virus obtained in Example 1 was inoculated into the treated CEF cells, and each dilution was repeated 4 times, and placed in 37 ° C, 5% CO 2 cultured in an incubator. When the CEF cells have obvious lesions, record the number of plaques that appear, calculate the average value of four replicate plaques for each dilution, and convert the virus plaque forming units (PFU) in the virus solution according to the number of plaques. The results showed that rFPV-VP1-VP2 and FPV titers were greater than 10 6 PFU / mL (see Table 1).

[0052] Table 1 Virus amplification titer

[0053]

[0054] b. Determination of Growth Curve of Recombinant Fowlpox Virus

[0055] Infect cells with recombinant fowlpox virus and fowlpox virus res...

Embodiment 3

[0070] Example 3: Detection of immune efficacy of recombinant fowlpox virus co-expressing chicken infectious anemia virus VP1 and VP2 genes

[0071] a. ELISA detects the change of CAV antibody titer in breeder chicken serum

[0072] 50 90-day-old SPF breeder chickens were inoculated with FPV and rFPV-VP1-VP2 wings, and the inoculation dose was 10 5 PFU / feather, the negative control group was inoculated with the same volume of normal saline in the same way. On the 0d, 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, and 63d after immunization, blood samples were taken from all test chickens under the wings and the serum was separated in time, and then the CAV antibody detection kit (purchased from IDEXX company, Antibody titer is negative lower than 999, and its detection CAV antibody highest titer value is 8861) detects the antibody titer change of test chicken, the result is as follows Figure 9 . The results showed that the anti-CAV antibody produced by the rFPV-VP1-VP2 recombinant...

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Abstract

The invention discloses a chicken infectious anemia virus VP1 and VP2 expression genetic recombination fowlpox virus living-vector vaccine. On the basis of a fowlpox virus transfer vector Psy681, recombination infectious clone Psy681-VP1-LacZ-VP2 is constructed, and in a CEF cell, pSY681-VP1-LacZ-VP2 and a FVP strain gene are subjected to homologous recombination through a homologous arm to obtainrFPV-VP1-VP2. A rFPV-VP1-VP2 viral strain can induce the body to generate an effective antibody for resisting CAV, and an immunoprotection effect is achieved; and after obtaining a high-level effective antibody for resisting CAV, offspring of chickens can resist invasion of CAV strong viruses well, and the immunoprotection effect is achieved.

Description

technical field [0001] The invention belongs to the field of animal vaccines, in particular to a recombinant fowl pox virus live vector vaccine expressing chicken infectious anemia virus VP1 and VP2 genes. Background technique [0002] Chicken infectious anemia (chicken infectious anemia, CIA) is an immunosuppressive disease caused by chicken infectious anemia virus (chicken anemia virus, CAV), which belongs to the circoviridae family and the genus Cyclovirus. Chicken is the main host of CAV. Chickens of all breeds and ages are susceptible, and chickens less than two weeks old are the most susceptible. With the growth of chicken age and the improvement of immune function, the susceptibility decreases. Serological Data show that all major poultry farming countries are prevalent in chicken infectious anemia virus. The main characteristic clinical symptoms of CIA are anemia, poor spirits, pale skin, loss of appetite, slowed weight gain, slow growth, and death of sick chickens ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/863A61K39/12A61K39/275A61K39/295A61P31/20C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/552A61K2039/70A61P31/20C07K14/005C12N7/00C12N15/86C12N2710/24021C12N2710/24034C12N2710/24043C12N2750/10022C12N2750/10034
Inventor 谢青梅周庆丰雷小亚李鸿鑫蔺文成陈伟国
Owner 广州金苗动保科技有限公司
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