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A recombinant fowlpox virus transfer vector expressing a fowl adenovirus serotype-4 fiber2 gene, a constructing method thereof and applications of the transfer vector

A technology of fowlpox virus and transfer vector, which is applied in the direction of virus/bacteriophage, application, virus, etc. It can solve the problems of poor immune effect, slow antibody production, and unsatisfactory effect, and achieve the effect of high cost and improved efficiency

Active Publication Date: 2017-12-15
WENS FOODSTUFF GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of vaccines, at present, some vaccine manufacturers have produced corresponding chicken embryo inactivated vaccines and cell-cultured inactivated vaccines, but the effect is not very satisfactory. Inactivated vaccines can only prevent the occurrence of the disease to a certain extent, and are used in immune prevention Some deficiencies existed at the time, such as the slow production of antibodies, the inability to distinguish between vaccine immunization and wild virus infection, and the inability to monitor the epidemic situation, the immune effect is poor, multiple immunizations are required, and the cost is increased. Therefore, it is necessary to develop more effective vaccines for chickens. Type 4 adenovirus for prevention and control
[0007] It can be seen that the existing technology still needs to be perfected

Method used

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  • A recombinant fowlpox virus transfer vector expressing a fowl adenovirus serotype-4 fiber2 gene, a constructing method thereof and applications of the transfer vector
  • A recombinant fowlpox virus transfer vector expressing a fowl adenovirus serotype-4 fiber2 gene, a constructing method thereof and applications of the transfer vector
  • A recombinant fowlpox virus transfer vector expressing a fowl adenovirus serotype-4 fiber2 gene, a constructing method thereof and applications of the transfer vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of recombinant fowlpox virus transfer vector expressing chicken type 4 adenovirus (FADV4) fiber2 gene

[0047] 1. Materials

[0048] 1.1 Virus strains and cells

[0049] Chicken pox virus quailization attenuated strain (CVCC AV1003) was purchased from Dahuanong Biotechnology Co., Ltd. of the company. Chicken type 4 adenovirus strain (F4) was collected from chicken farms and stored in the laboratory. Chicken embryo fibroblasts (CEF) were used for warming SPF Chicken Embryo Preparation of Shi Company.

[0050] 1.2 Plasmids and strains

[0051] Competent JM109 was purchased from Treasure Biotech Company, the plasmid pMD22-TYB containing the multiple cloning site of fowlpox virus early and late promoter LP2EP2, the selection gene lacz gene and the genome TYB non-replication region fragment of fowlpox virus for homologous recombination -lacz was built for this lab.

[0052] 1.3 Main reagents

[0053] High-fidelity enzyme PrimeSTAR, restriction enz...

Embodiment 2

[0062] Example 2 Verifying the effect of the transfer vector pMD22-TYB-lacz-F4 in Example 1

[0063] Identification of Recombinant Fowlpox Virus

[0064] Extract the transfer vector pMD22-TYB-lacz-F4 in Example 1 according to the instructions of the plasmid kit, and prepare CEF cells. When the cells grow to about 80%, inoculate the virus liquid of the chickenpox vaccine strain, incubate at 37°C for 2 hours, and wash After the cells, the transfer vector pMD22-TYB-lacz-F4 was transfected according to the instructions of the Lipofectamine 2000 reagent, and the cells were collected when 80% of the lesions occurred in the cells, and the cells were frozen and thawed three times, and the blue and white spots were screened with X-gal, such as image 3 As shown, pick the blue spot, freeze and thaw three times repeatedly, extract DNA, and use primers F4-fiber2-F, F4-fiber2-R for PCR detection, the results are as follows Figure 4 As shown, a 1440bp fragment was amplified, proving that ...

Embodiment 3

[0067] Example 3 Preparation of anti-chicken type 4 adenovirus FPV vector vaccine

[0068]Co-transfect chicken embryo fibroblasts (CEF) with the transfer vector pMD22-TYB-lacz-F4 and fowlpox virus, screen blue and white spots with X-gal, pick positive blue spots, and continue to subculture until all spots are blue Inoculate the selected positive coeruleus cells on the monolayer of CEF cells, incubate at 37°C in a 5% CO2 incubator for 2 hours, then continue to screen and purify the coeruleus coeruleus, and repeat this process for multiple generations Purification until the plaques appearing in all lesions are blue spots, then the purification is completed; the purified virus liquid is inoculated on CEF cells for passage, and the virus liquid is collected. The virus liquid can be used to produce anti-chicken type 4 adenovirus recombinant fowlpox virus transfer vector vaccine.

[0069] Laboratory verification of efficacy of FPV vector vaccine against chicken adenovirus type 4

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Abstract

The invention belongs to the technical field of biology, and mainly relates to a recombinant fowlpox virus transfer vector expressing a fowl adenovirus serotype-4 (FADV4) fiber2 gene, a constructing method thereof and applications of the transfer vector. A pMD19T-Simple vector is adopted as a base of the transfer vector. The FADV4 fiber2 gene, a lacz gene, and fowlpox virus genome replicated non-essential fragments which are LTYB and RTYB used for homologous recombination are inserted in a TA cloning site. The method includes constructing a plasmid pMD-TYB; constructing a plasmid pMD22; constructing a plasmid pMD22-lacz; constructing an intermediate vector pMD22-TYB-lacz; amplifying the FADV4 fiber2 gene; constructing a recombinant fowlpox virus transfer vector pMD22-TYB-lacz-F4; subjecting a chicken embryo Fibroblast to cotransfection with the transfer vector pMD22-TYB-lacz-F4 and a fowlpox virus; performing identification to select a positive product; performing subculture continuously; and identifying expression effects of the recombinant fowlpox virus. The recombinant fowlpox virus transfer vector constructed by the method lays a foundation for development of an efficient recombinant fowlpox virus genetic engineering living-vector vaccine expressing the fowl adenovirus serotype-4.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to a recombinant fowlpox virus transfer vector expressing the fiber2 gene of chicken type 4 adenovirus (FADV4) and its construction method and application. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) is a double-stranded DNA virus without an envelope. The avian adenoviruses with different antigens can be divided into 3 groups: Ⅰ, Ⅱ and Ⅲ. Group I avian adenoviruses are mainly obtained from chickens, turkeys, geese and other poultry, and are divided into five species (A-E). The viruses in each species are further divided into 12 serotypes based on the results of cross-neutralization tests ( FAdV1-12). Avian adenovirus C includes avian adenovirus types 4 and 10 (FAdV-4, FAdV-10), and its main clinical and pathological syndromes are hydropericardium syndrome and inclusion body hepatitis. [0003] The capsid of avian adenovirus is mainly composed of hexon a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/863C12N15/66C12N15/34A61K39/235A61P31/14
CPCA61K39/12A61K2039/5256A61K2039/552C07K14/005C12N15/86C12N2710/10222C12N2710/10234C12N2710/24043
Inventor 林丽苗周庆丰李群辉招丽婵余国莲李薇
Owner WENS FOODSTUFF GRP CO LTD
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