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Construction method of PEDV (porcine epidemic diarrhea virus) M gene and S1 gene series recombinant plasmid

A technology for recombinant plasmids and construction methods, which is applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., and can solve the problems of poor immune effect of PED vaccines, etc.

Active Publication Date: 2017-09-22
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the mutation of the PEDV-S gene may be the reason for the poor immune effect of the PED vaccine

Method used

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  • Construction method of PEDV (porcine epidemic diarrhea virus) M gene and S1 gene series recombinant plasmid
  • Construction method of PEDV (porcine epidemic diarrhea virus) M gene and S1 gene series recombinant plasmid
  • Construction method of PEDV (porcine epidemic diarrhea virus) M gene and S1 gene series recombinant plasmid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1 Materials and methods

[0028] 1.1 Viruses and expression vectors

[0029] Expression vector: pET-32a(+) (purchased from Promega Beijing Biotechnology Co., Ltd.); Cloning and expression host bacteria: DH5α and E.coli BL 21 (DE3) competent cells (purchased from Promega Beijing Biotechnology Co., Ltd.).

[0030] 1.2 Main reagents and instruments

[0031] Main instruments: DL777 PCR instrument (Beijing Donglin Biotechnology Co., Ltd.), TG16-W micro high-speed centrifuge (Weirkang Xiangying Centrifuge Co., Ltd.), ZHWY-103D constant temperature culture oscillator (Zhicheng Analytical Instrument Manufacturing Co., Ltd.), DYY16D Electrophoresis instrument (Beijing Liuyi Biotechnology Co., Ltd.), Bio-Rad gel imaging analysis system (Bio-rad, USA).

[0032] Main reagents: 2000bpDNALadder (Tiangen Biochemical Technology Co., Ltd.), viral RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), reverse transcription kit (Promega Beijing Biotechnology Co., Ltd.)...

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Abstract

The invention provides a construction method of PEDV (porcine epidemic diarrhea virus) M gene and S1 gene series recombinant plasmid; the construction method comprises: selecting genome of an epidemic PEDV strain as a template, designing specific primers respectively for main antigen sites of S1 gene and M gene, using PET-32a as an expression vector to design a specific restriction enzyme cutting site, and constructing the S1 gene and M gene series recombinant plasmid. The S1 gene and M gene series recombinant plasmid is capable of recombine-expressing main antigen sites of S1 protein and M protein in vitro at the same time, and important basis can be provided for the research and development of PEDV genetically-engineered vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a PEDV M gene and an S1 gene tandem recombination plasmid. Background technique [0002] Porcine epidemic diarrhea is an intestinal infectious disease caused by epidemic diarrhea virus (PEDV) infection, with loss of appetite, vomiting, diarrhea and dehydration as the main clinical symptoms. In recent years, due to the mutation of the virus strain, the disease has caused great harm to suckling piglets, and its infection rate can reach 100%, which has brought a great impact on both the pig industry and farmers in our country. There is currently no effective drug for the treatment of this disease. [0003] PEDV belongs to the genus Coronaviridae and contains 5 open reading frames (ORFs), encoding 4 main structural proteins. The S protein is a spike protein on the surface of the virus particle, which is mainly involved in the adsorption and fusion ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C12N15/70C12N15/11C12N15/66
CPCC07K14/005C12N2770/20022C12N2770/20051
Inventor 王全溪刘梦茜陈媛
Owner FUJIAN AGRI & FORESTRY UNIV