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Method for rapidly screening recombinant fowlpox virus by means of CRISPR/Cas9 system and application thereof

A fowlpox virus, rapid technology, applied in the biological field, can solve problems such as long production cycle, achieve the effects of low cost, reduced production cost and high efficiency

Active Publication Date: 2017-12-08
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of constructing recombinant fowlpox virus, the currently used method is to use liposome or electroporation to co-transfect the plasmid containing the expression foreign gene with the fowlpox virus, and then carry out plaque detection by reporter groups EGFP and lacz. Screening, it takes more than ten generations of screening to get pure recombinant fowlpox virus, and the production cycle is long

Method used

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  • Method for rapidly screening recombinant fowlpox virus by means of CRISPR/Cas9 system and application thereof
  • Method for rapidly screening recombinant fowlpox virus by means of CRISPR/Cas9 system and application thereof
  • Method for rapidly screening recombinant fowlpox virus by means of CRISPR/Cas9 system and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] 1. Materials

[0042] 1.1 Virus strains and cells

[0043] Chicken pox virus quailization attenuated strain (CVCC AV1003) was purchased from Dahuanong Biotechnology Company of the company, chicken type 4 adenovirus strain was isolated and preserved from chicken farms, and chicken embryo fibroblasts (CEF) were obtained from Wen’s SPF chicken Embryo preparation.

[0044] 1.2 Plasmids and strains

[0045] Competent JM109 was purchased from Treasure Biotech Co., Ltd., and the transfer plasmid pMD22-TYB-lacz-F4 expressing FADV4 fiber2 recombinant fowlpox virus (rFPV-FADV4fiber2) was self-constructed. For the construction process, see figure 1 .

[0046] 1.3 Main Reagents

[0047] CRISPR / Cas9 kit, IPTG, X-gal were purchased from TAKARA company, DNA recovery kit and plasmid extraction kit were purchased from OMEGA company, DNA extraction kit was purchased from Axygen company, fetal calf serum (FCS) and DMEM culture The base was purchased from Thermo Company, and the trans...

Embodiment 3

[0084] Example 3 Application of the method of the present invention for screening recombinant fowlpox virus in the preparation of anti-chicken type 4 adenovirus FPV vector vaccine

[0085] according to figure 1 The rFPV-FADV4 fiber2 transfer vector was constructed as shown, and the constructed vector was co-transfected with the CRISPR / Cas9 plasmid expressing sgRNA constructed above at a ratio of 1:1 to CEF cells infected with fowlpox virus, cultured, and passed through X-GAL Blue-white screening was carried out, and the purified rFPV-FADV4 fiber2 virus was obtained after four generations of screening. It was passaged on CEF cells several times, and the virus liquid was collected to provide the virus liquid for the preparation of anti-chicken adenovirus type 4 FPV vector vaccine. , the collected virus liquid is operated according to the conventional operation preparation method, and the anti-chicken type 4 adenovirus FPV vector vaccine is prepared.

[0086] Verification of the...

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Abstract

The invention relates to the field of biotechnology, in particular to a method for rapidly screening the recombinant fowlpox virus by means of a CRISPR / Cas9 system and application thereof. The method comprises the steps of designing an sgRNA (short guide RNA) double-stranded oligonucleotide sequence of a target gene, ligating the sequence with a linearized plasmid vector to obtain an sgRNA expression vector, co-transfecting chicken embryo fibroblasts (CEF) with the sgRNA expression vector, recombinant fowlpox virus plasmids expressing a foreign gene and the fowlpox virus, and conducting purification and verifying the purification effect. By means of the CRISPR / Cas9 method, the number of recombinant fowlpox virus screening generations is reduced to 3-4, the efficiency of vaccine production is greatly improved, operation is easy, and the production cost is reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly screening recombinant fowlpox virus through a CRISPR / Cas9 system and its application. Background technique [0002] The non-replication region of the highly conserved region of the fowlpox virus genome can integrate foreign DNA, which has become a common expression vector and has been widely used in the fields of virus molecular biology and vector vaccine production. The fowlpox virus vector vaccine can not only prevent the occurrence of fowlpox, but also can induce the expression of the inserted gene to improve the immune resistance of the corresponding pathogen by inserting the protective antigen gene of other pathogens. Since the foreign gene expressed by the recombinant fowlpox virus is expressed with the replication of the carrier virus in the body, compared with the inactivated vaccine, the immunization dose is small, the vaccination method is simple, and o...

Claims

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Application Information

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IPC IPC(8): C12N15/863C12N15/90C12N7/01A61K39/295A61K39/275A61K39/235A61P31/20
CPCA61K39/12A61K2039/5256A61K2039/552A61K2039/70C12N7/00C12N15/86C12N15/902C12N2710/10234C12N2710/24021C12N2710/24034C12N2710/24043
Inventor 周庆丰林丽苗李群辉余国莲李薇杜云平
Owner WENS FOOD GRP CO LTD
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