Construction Of A Vero Cell Line Deficient Of The Type ? Interferon Gene By CRISPR/Cas9-mediated Gene Targeting Technology And Proliferating Features Of PEDV In It

Porcine epidemic diarrhea is a viral infectious disease caused by porcine epidemic diarrhea virus.PEDV belongs to the coronavirus family.It is a single enveloped RNA virus.The piglets in 1 weeks of age are usually infected with PEDV after persistent diarrhea and vomiting after 3-4 days of dehydration.The older pigs usually recover after 1 weeks.Diarrhea and diarrhea may occur after the infection of fattening pigs and sows,but their mental state is poor,and the spirit is depressed,anorexia,supine and thinning.The mortality of piglets infected with 7 days old is very high.Pigs infected with porcine epidemic diarrhea virus will delay the time of slaughter and increase the cost of raising pigs.With the outbreak of PEDV in many countries in recent years,it has brought great harm to the pig industry worldwide.Scholars in various countries are studying the culture conditions of PEDV in vitro so as to improve the titer of the virus.After the PEDV infected cells,the target cells secrete interferon to protect the cells from the virus infected by the virus,which leads to the low virus titer in the culture of PEDV.After the virus infected cells,the target cells release interferon.The released interferon will bind to the IFN receptor on the surface of the downstream cells,activate the signal transduction pathway in the cell,and regulate the cell growth and differentiation by activating the protein molecules on the signal transduction pathways in multiple cells,making the downstream cells free from the virus.Infection,so as to achieve the purpose of antiviral.Interferon itself does not play a direct role in killing or neutralizing viruses.In order to improve the reproductive titer of PEDV,by knocking off the I type interferon receptor gene on Vero cells,the PEDV continuum was cultured in the external generation,and the titer of the virus was improved,which laid a certain foundation for the development of the next vaccine.This article is divided into the following four parts:1.Construction of plasmids pX330-IFNR-sg RNA:IFNAR1-F and IFNAR1-R were annealed to form double stranded DNA and reclaimed linear pX330 plasmid to form a specific targeting vector pX330-IFNR-sg RNA,the product was transformed into DH5 alpha,and the plasmid was selected for single colony extraction plasmid and identified,and the plasmid was extracted from the correct plasmid and then extracted without endotoxin plasmids.2.The establishment of cell lines:The constructed plasmid pX330-IFNR-sg RNA and the plasmid with resistance marker were transfected into normal Vero cells,and 10% serum containing 10% DMEM were cultured with G418 concentration of 200 mu g/mL,400 micron g/mL and 500 u g/mL respectively.Finally,the monoclonal cells were selected and then cultured.Extraction of genomic DNA from monoclonal cell lines and identification of PCR.3.The effect of I type interferon receptor deletion on the value of PEDV added value: 0.05 MOI PEDV GH strains infected WT Vero and IFNAR1-/-Vero cells,respectively,in 24,48,72,96 hpi,respectively,to determine the virus titer and the use of fluorescence quantitative PCR to determine the copy number of the virus genome.The results showed that from 24 to 72 HPI,the titer and virus copy number of GH strain on IFNAR1-/-Vero cells were significantly higher than that of WT Vero cells,but the titer and copy number of GH strain on IFNAR1-/-Vero cells were lower than that of WT Vero cells in 96 HPI,and the values were 0.054 > 0.05,and the difference was not significant.4.In this study,the Vero cells with I type interferon receptor deletion were successfully constructed.The proliferation of PEDV in the cells increased significantly,laying the foundation for the development of higher titer PEDV vaccine.