Construction method of recombinant coatpox virus used for expressing foot and mouth disease virus empty capsid

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...

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Problems solved by technology

The three are indistinguishable morphologically, physicochemically, and serologically, and can only be differentiated by genome

[0016] Homologous recombination also has disadvantages and deficiencies: First, the probability of homologous recombination is small. If a large fragment of foreign DNA is inserted into the viral genome, the efficiency of homologous recombination will be greatly reduced. Obtain purified recombinant virus; another disadvantage of homologous recombination is that it needs to go through complicated steps such as constructing an intermediate plasmid (transfer vector) and amplifying it in the host cell. If the target gene fragment is relatively large or has a special structure, it may cause Gene deletion or rearrangement occurs inside the recombinant plasmid, and when the target gene is toxic to the host bacteria, this strategy is more difficult to work

However, the decline in virulence will affect the invasiveness of the virus in the body, so the expression level of foreign genes will also decrease accordingly

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