Reverse genetic system for rescuing PEDV (porcine epidemic diarrhea virus) ZJU/G2/2013 strain based on DNA (deoxyribonucleic acid) plasmid transfection

Abstract

The invention discloses a reverse genetic system for saving a PEDV (porcine epidemic diarrhea virus) ZJU/G2/2013 strain based on DNA (deoxyribonucleic acid) plasmid transfection. The kit comprises a recombinant expression vector containing whole genome cDNA of a porcine epidemic diarrhea virus G2 gene subtype ZJU/G2/2013 strain, wherein an ORF3 region is replaced with a GLuc reporter gene; the kitcomprises a helper plasmid of an N gene of a porcine epidemic diarrhea virus G2 gene subtype ZJU/G2/2013 strain, and the GenBank number of the whole genome sequence of the porcine epidemic diarrhea virus G2 gene subtype ZJU/G2/2013 strain is KU558701.1. The infectious clone constructed by the invention has a GLuc reporter gene, and the rescued recombinant virus can generate secretory Gaussia luciferase when infecting animals and cells, is very easy to detect, and has very high application value in experimental research aspects such as virus toxicity analysis and the like. Meanwhile, as the ORF3 region is replaced with the GLuc reporter gene, the recombinant virus rescued by the system can be prepared into a novel marker vaccine, the vaccine strain immunity and wild strain infection can bequickly distinguished by detecting whether Gaussian luciferase exists in pig serum or not, and the practical value is very high.

Description

technical field [0001] The invention relates to the technical field of coronavirus reverse genetics, in particular to a reverse genetic system for rescuing porcine epidemic diarrhea virus G2 gene subtype ZJU / G2 / 2013 strain based on DNA plasmid transfection. Background technique [0002] Porcine epidemic diarrhea is an acute and highly contagious enteric infectious disease of pigs, which is caused by porcine epidemic diarrhea virus (PEDV). The disease is characterized by acute enteritis, vomiting, watery diarrhea and loss of appetite. Pigs of all ages are susceptible, but newborn piglets are the most serious, and its morbidity and mortality can reach 90%-100%. The disease was first discovered in Europe and is now widely prevalent in many Asian countries including China, South Korea, and the Philippines. It has become one of the main infectious diseases that seriously endanger the pig industry. At present, the PEDV prevalent in Asia and America is mainly the G2 gene subtype ...

AI Technical Summary

Problems solved by technology

[0008] Aiming at the deficiencies in the prior art, the present invention establishes a reverse genetic system for rescuing the PEDVZJU / G2 / 2013 strain based on DNA plasmid transfection, which overcomes the cDNA clone of the coronavirus partial replicase gene in the prior art in bacteria It overcomes the problem of low efficiency of traditional enzyme digestion and ligation, and also overcomes the problem of transcript heterogeneity caused by in vitro transcription.

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