A Reverse Genetics System for Rescuing pedv ZJU/G2/2013 Strain Based on DNA Plasmid Transfection
A technology of reverse genetics and plasmid transfection, which is applied in the direction of recombinant DNA technology, positive-sense single-stranded RNA virus, and microbial detection/testing, and can solve the problems of unstable preservation, heterogeneity of transcripts, and low efficiency of enzyme-cut ligation, etc. problem, to achieve strong application value, high sensitivity, and speed up the effect of the determination scheme
Active Publication Date: 2022-03-25
ZHEJIANG UNIV
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[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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Embodiment 1
[0043] 1. Amplification of the target fragment of PEDV-G2-ZJU-GLuc full-length infectious clone
[0044] According to the gene sequence of porcine epidemic diarrhea virus G2 subtype ZJU / G2 / 2013 strain published by the inventor on Genbank (Genbank number is KU558701.1, the gene structure is as follows: figure 1 ), the full length of the viral genome is 28038nt. Take the cell-infected supernatant sample of the stably passaged strain ZJU / G2 / 2013 strain (source: Zhejiang virulent strain isolated by the inventor in 2013), extract the RNA by the Trizol method and reverse-transcribe to obtain cDNA, using the method provided by the present invention. 5 pairs of primers (primer sequences are shown in Table 2), the corresponding 5 full-length genome fragments were obtained by amplification, cloned through the pCR-Blunt vector, picked 3 monoclonal colonies, extracted plasmids for sequencing and spliced the sequencing results to determine ZJU / The full-length genome sequence of G2 / 201...
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The invention discloses a reverse genetic system for rescuing PEDV ZJU / G2 / 2013 strain based on DNA plasmid transfection, comprising: recombinant expression of the whole genome cDNA of ZJU / G2 / 2013 strain containing porcine epidemic diarrhea virus G2 gene subtype Vector, wherein the ORF3 region is replaced by a GLuc reporter gene; a helper plasmid comprising the N gene of the porcine epidemic diarrhea virus G2 gene subtype ZJU / G2 / 2013 strain, said porcine epidemic diarrhea virus G2 gene subtype ZJU / G2 / 2013 The GenBank number of the whole genome sequence of the strain is KU558701.1. The infectious clone constructed by the present invention has a GLuc reporter gene, and the rescued recombinant virus will produce secreted Gaussia luciferase when infecting animals and cells, which is very easy to detect and has strong applications in experimental research such as virus virulence analysis value. At the same time, because the ORF3 region is replaced by the GLuc reporter gene, the recombinant virus rescued by this system can be made into a new type of labeled vaccine. By detecting whether there is Gaussia luciferase in pig serum, it can quickly distinguish between vaccine strain immunity and wild strain infection, which is of practical value 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, highly contagious intestinal infectious disease in pigs, caused by porcine epidemic diarrhea virus (PEDV). The disease is characterized by acute enteritis, vomiting, watery diarrhea, and decreased appetite. Pigs of all ages are susceptible, but newborn piglets are the most serious, and the morbidity and mortality can reach 90%-100%. The disease was first discovered in Europe and is now widespread in many Asian countries, including China, South Korea, and the Philippines. It has become one of the major infectious diseases that seriously endanger the pig industry. At present, the prevalent PEDV strains in Asia and America are mainly G2 subtype strains, a...
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IPC IPC(8): C12N15/85C12N15/65C12N15/50C12N7/01C12Q1/66G01N33/569
CPCC12N15/85C12N15/65C12N7/00C12Q1/66G01N33/56983C07K14/005C12N2770/20021C12N2770/20022
Inventor 黄耀伟王斌于佳琦
Owner ZHEJIANG UNIV