Prokaryotic fusion expression vector for expressing M+N double genes of PEDV (porcine epidemic diarrhea virus) and application

Abstract

The invention belongs to the field of molecular biology and particularly relates to a prokaryotic fusion expression vector for expressing M+N double genes of PEDV (porcine epidemic diarrhea virus). The prokaryotic fusion expression vector for expressing M+N double genes of the PEDV is constructed with a seamless cloning technology on the basis of M and N gene fragments of the PEDV and a Blunt-E2 expression vector, and an application of the prokaryotic fusion expression vector to recombined expression of genetic engineering protein is provided. The purified M gene fragment of the PEDV is linked directly to a Blunt-E2 prokaryotic fusion expression vector of a mammal instead of being linked to a monoclonal vector PMD19-T, then a plasmid M+Blunt-E2 is seamlessly spliced with the N gene fragment with a homologous recombination technology, and the prokaryotic fusion expression vector for expressing M+N double genes is obtained. The M+Blunt-E2 plasmid is accurately sequenced by verifying with a PCR technology and transfected into cells, and successful expression is realized. Therefore, preparation of the prokaryotic fusion expression vector has the characteristics of simple operation steps, short reaction time and the like as compared with other prokaryotic fusion expression technologies.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a prokaryotic fusion expression vector expressing M+N double genes of PEDV and application thereof. Background technique [0002] Porcine epidemic diarrhea (PED) is a viral disease of the digestive tract characterized by vomiting, diarrhea, dehydration and death in pigs caused by porcine epidemic diarrhea virus (PEDV). The disease was first reported in the UK in 1971, and our country confirmed the existence of the disease in 1984. Since the second half of 2010, due to the deletion, insertion and mutation of the traditional PEDV gene, the pathogenicity of the disease to pigs has increased, causing a large number of pigs to die and suffer heavy losses. At present, there are reports of mutated PEDV in most pig-raising countries in the world, especially in China, South Korea, Japan, Thailand and the United States, which have caused huge economic losses to the pig industr...

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Problems solved by technology

At present, single genes such as N, S and M gene fragments of PEDV have been seen to express single-gene proteins through prokaryotic expression methods, but no M and N gene fragments of PEDV have been seen, using pEASY-Blunt-E2 prokaryotic expression vector , Through seamless cloning technology, a double-gene prokaryotic expression vector of PEDV M+Blunt-E2+N was constructed

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