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A recombinant fowlpox virus transfer vector expressing a duck adenovirus serotype-2 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., to overcome the effect of easy to disperse the virus and improve the construction efficiency

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

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Problems solved by technology

[0005] 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 duck adenovirus serotype-2 fiber2 gene, a constructing method thereof and applications of the transfer vector
  • A recombinant fowlpox virus transfer vector expressing a duck adenovirus serotype-2 fiber2 gene, a constructing method thereof and applications of the transfer vector
  • A recombinant fowlpox virus transfer vector expressing a duck adenovirus serotype-2 fiber2 gene, a constructing method thereof and applications of the transfer vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Materials

[0036] 1.1 Virus strains and cells

[0037] The quailized attenuated strain of chickenpox virus (CVCCAV1003) was purchased from Dahuanong Biotechnology Co., Ltd., and the duck adenovirus type 2 was isolated from a duck farm of the company (the first strain of duck adenovirus type 2 in China was identified by metagenomics. Chinese Journal of Preventive Veterinary Medicine, 2015, 37:903-907), chicken embryo fibroblasts were prepared from SPF chicken embryos of Wen's company.

[0038] 1.2 Plasmids and strains

[0039] Plasmids pMD19T-Simple, pGuide-it-ZsGreen1, and competent JM109 were purchased from Bao Biological Company, and plasmid pSV-β-Galactosidase Control Vector was purchased from Promega Company, containing the multiple cloning sites of the early and late promoters of fowlpox virus LP2EP2 and P11 promoters The plasmid pMD22 of the dot sequence was synthesized by Suzhou Shengxin Biotechnology Company.

[0040] 1.3 Main Reagents

[0041] High-fide...

Embodiment 2

[0070] Validate the effect of the transfer vector pMD22-TYB-lacz-DFB2 in Example 1

[0071] 1. Verification of the induced expression effect of the early and late promoters containing fowlpox virus synthesized in the plasmid pMD22-TYB-lacz

[0072]According to the green fluorescent group ZsGreen sequence in the plasmid pGuide-it-ZsGreen1 purchased from TAKARA company, primers pGREEN-F and pGREEN-R (see Table 1) were designed to amplify the ZsGreen gene, and the amplified fragment size was 696bp (SEQ ID NO: 7). Insert it into the SmaI site of the plasmid pMD22-TYB-lacz by infusion method to obtain the plasmid pMD22-TYB-lacz-G; extract and transfer the plasmid pMD22-TYB-lacz according to the instructions of the Life Company Plasmid Midiprep Kit (PureLink HiPure PlasmidMidiprep Kit) -G, prepare CEF cells, when the cells grow to a single layer, inoculate the quailized attenuated strain (CVCCAV1003) fowl pox virus vaccine (passed 10 generations), incubate at 37°C for 2 hours in a 5...

Embodiment 3

[0075] Example 3 Preparation of Recombinant Fowlpox Virus Transfer Vector Vaccine against Duck Adenovirus Type 2

[0076] Utilize the carrier of Example 1 to prepare the recombinant fowlpox virus transfer vector vaccine against duck type 2 adenovirus, the specific operations are:

[0077] (1) Extract the transfer vector pMD22-TYB-lacz-DFB2 in Example 1 according to the instructions of Life Company’s Plasmid Midiprep Kit (PureLink HiPure Plasmid Midiprep Kit) to prepare CEF cells (chicken embryo fibroblasts). When the cells grow to In a single layer, inoculate the quailized attenuated strain (CVCCAV1003) fowl pox virus vaccine (passed 10 generations), incubate at 37°C in a 5% CO2 incubator for 2 hours, and transfect the pMD22-TYB-lacz-DFB2 plasmid according to the instructions of Lipofectamine 2000 from Life Company , Collect the virus liquid at about 72 hours, freeze and thaw three times repeatedly; dilute the above virus liquid by 10-1 to 10-3 times, inoculate on CEF cells, i...

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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 duck adenovirus serotype-2 (DADV2) fiber2 gene, a constructing method thereof and applications of the transfer vector. According to the transfer vector, a DADV2 fiber2 gene promoted by a fowlpox-virus-containing early-late promoter LP2EP2, a lacz gene promoted by a P11 promoter, and fowlpox virus genome replicated non-essential fragments which are LTYB and RTYB used for homologous recombination are inserted in a TA cloning site of a pMD19T-Simple vector. The method includes constructing a plasmid pMD-TYB; constructing a plasmid pMD22; constructing a plasmid pMD22-lacz; constructing an intermediate vector pMD22-TYB-lacz; constructing the transfer vector pMD22-TYB-lacz-DFB2; and subjecting the transfer vector pMD22-TYB-lacz-DFB2 to effect verification. 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 duck adenovirus serotype-2.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to a recombinant fowlpox virus transfer vector expressing duck type 2 adenovirus (DADV2) fiber2 gene, a construction method and application thereof. Background technique [0002] Duck Adenovirus 2 (Duck Adenovirus 2) belongs to the family Adenoviridae and is a linear double-stranded DNA virus with a diameter of 70-90nm. Wrapped in the capsid, the capsid is composed of 252 capsicles, of which 240 are hexons and 12 are penton bases, each base is combined with 2 fiber protrusions, and these two fiber protrusions are fibers Proteins are encoded by fiber1 and fiber2 genes, respectively. The length of fibrin is related to the antigenicity of the virus, with adenovirus type and subgenus-specific epitopes, and is the main protein that stimulates the host to produce antibodies. Among them, fiber2 can induce the body to produce the main type-specific neutralizing antibody, which is necessa...

Claims

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Application Information

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IPC IPC(8): C12N15/863C12N15/66C12N15/34A61K39/235A61P31/20
CPCA61K39/12C07K14/005C12N15/86C12N2710/10222C12N2710/10234C12N2710/24043
Inventor 林丽苗周庆丰李群辉招丽婵李薇余国莲杜云平
Owner WENS FOOD GRP CO LTD
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