A method for constructing recombinant goat pox virus expressing empty capsid of foot-and-mouth disease virus

A technology of foot-and-mouth disease virus and goat pox virus, which is applied in the direction of virus/bacteriophage, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of affecting virus invasiveness, reducing the expression level of foreign genes, gene deletion, etc.

A technology of foot-and-mouth disease virus and goat pox virus, which is applied in the direction of virus/bacteriophage, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of affecting virus invasiveness, reducing the expression level of foreign genes, gene deletion, etc.

CN103343110BInactive Publication Date: 2014-10-08新疆维吾尔自治区畜牧科学院兽医研究所
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  • A method for constructing recombinant goat pox virus expressing empty capsid of foot-and-mouth disease virus
  • A method for constructing recombinant goat pox virus expressing empty capsid of foot-and-mouth disease virus

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Experimental program
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Embodiment

[0047] A kind of construction method of the recombinant goat pox virus expressing foot-and-mouth disease virus empty capsid of the present invention, concrete operation is carried out according to the following steps:

[0048] 1. Cells, Strains and Vectors

[0049] 1.1 MDBK cells are preserved and prepared by our laboratory; the recipient bacteria E.coli are preserved by our laboratory;

[0050] 1.2 Goat pox virus vaccine (GTPV CVCC AV41 strain) was purchased from Xinjiang Tiankang Animal Husbandry Biotechnology Co., Ltd., and the virus TCID was determined 50 1×10 -0.5 / 0.1mL;

[0051] 1.3 Plasmid pCI was purchased from Promega Company; foot-and-mouth disease virus 3C protease 9-site mutant gene cloning plasmid p9m3C plasmid, foot-and-mouth disease virus structural protein gene cloning plasmid pmP1-2A (Js / 05), constructed and preserved by the applicant;

[0052] 2. Construction of cloning plasmid p9m3C of foot-and-mouth disease virus 3C protease 9-site mutant:

[0053] 2.1...

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Abstract

The invention relates to a construction method of recombinant coatpox virus used for expressing foot and mouth disease virus empty capsid. The method comprises the following steps of: constructing transfer plasmids on the basis of forward and reverse homologous arms; inserting a foot and mouth disease virus protein precursor gene P1-2A, a vaccinia virus back-to-back promoter P7.5-P11, a hypoxanthine-guanine phosphoribosyltransferase gene, a foot and mouth disease virus internal ribosome entry site sequence and a foot and mouth disease virus 3C protease 9-site mutant gene to the transfer plasmids in sequence; co-transfecting an MDBK (Madin-darby Bovine Kidney) cell through the transfer plasmids and the coatpox virus; and then inserting an exogenous gene between the forward and reverse homologous arms in a homologous recombination way. By adopting the construction method, the screened recombinant coatpox virus can exist stably and can be used for expressing the foot and mouth disease virus empty capsid.

Description

technical field [0001] The invention relates to a method for constructing a recombinant goat pox virus expressing empty capsid of foot-and-mouth disease virus. Background technique [0002] Since vaccinia virus was successfully used to express foreign proteins in 1982, the research on recombinant live vector vaccines has made great progress. The TK gene of herpes simplex virus was co-transfected into eukaryotic cells for homologous recombination in vivo to construct recombinant vaccinia virus. In 1990, the recombinant fowlpox virus expressing the F gene was registered in the United States; in 1994, the United States approved the production of recombinant vaccines expressing the HN and F protein genes of Newcastle disease virus, becoming the first commercial live vector vaccine; in 1995, the United States Department of Agriculture officially approved Commercialization of recombinant fowl pox virus live vector vaccine VAX FP-N. Preliminary research results have shown that the...

Claims

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Application Information

Patent Timeline
08 Oct 2014
Publication
CN103343110B
IPC
C12N7/01; C12N15/63
Inventors
马文戈; 魏婕