Construction Of Viperin-partial-deficient PK-15 Cells And Its Application On Culture PCV2

Porcine circovirus type 2(PCV2)has been shown to be associated with weaning piglet syndrome and other clinical disorders,which is including porcine reproductive disorders,dermatitis and nephrotic syndrome,necrotizing tracheitis,fetal myocarditis,and congenital tremor.Vaccination has been the main way of PCV2 prevention and control.At present,PCV2 vaccines are mainly inactivated vaccines and subunited vaccines.PCV2 subunit vaccines areproduced by means of genetic engineering,which generate recombinant Cap protein in large quantities.However,subunit vaccines also havemany problems,such as high production cost,difficult to purify,and endotoxin problem as wel.Most inactivated vaccines are made of inactivated whole viruses which are derived from pk-15 cell culture,but the current culture process can't solve the problems of slow virus proliferation and low virus content,which seriously limit the production level of inactivated vaccine.In order to reduce the antiviral response of pk-15 cells in the production of PCV2 virus antigen and improve the low titer of virus proliferation,this study established the CRISPR/Cas9 based gene editing method to knock out viperin gene,one of the key antiviral genes from the genome of pk-15 cell,and explored the efficient proliferation mode of virus in viperin-deficient cells.Viperin protein belongs to type I interferon(IFN-I),which has been shown to have a broad antiviral effect.It is located in the endoplasmic reticulum and can interfere with viral particle replication and inhibit virus reproduction.In order to further study the role of Viperin protein in pig cells,primers for three exons of pig Viperin gene were designed according to the pig viperin sequence downloaded from NCBI.The PCR products which length were 529 bp,385bp and 639 bp,respectively were sent for sequencing.After that,g RNAs(single guide RNAs)was designed according to the three amplified exon fragments.g RNA was inserted in to the p X459 plasmid by molecular cloning method.After sequencing,p X459 recombinant vector was transfected into pk-15 cells with optimized liposomes transfectionmethod.PK-15 cells with viperin gene deletion were screened by adding purinomycin into transfected cell culture medium.The deletion of viperin gene was identified by i PCR amplification and sequencing.After subcloning,two cell clones of viperin showed different deletion in the viperin genome sequence were named pk-15-vip1 cell and pk-15-vip2 cell.The CRISPR/Cas9 technology can accurate and efficient edit pk-15 cell genes,with an editing efficiency of up to 70% assisted by antibiotic pressure screening.Through the observation of cell growth state and growth curve,it was found that the cell lines with gene deletion had stronger vitality and faster growth rate.In the PCV2 infection test,it was found that the viral titer of PCV2 cultured in pk-15-vip1 cells was significantly higher than that of wild-type cell lines,while the viral proliferation titer of pk-15-vip2 cells was not significantly different from that of pk-15 wild-type cells.The Cap genes of PCV2 virus cultured in pk-15-vip1 cells and pk-15 wild-type cells were sequenced and compared respectively.It was found that the deletion of viperin gene had no effect on the Cap genes of PCV2 virus,so the PCV2 immunogenicity of proliferation on pk-15-vip2 cells was not changed.In this experiment,viperin-deficient pk-15 cells were successfully constructed by CRISPR/Cas9 technology,and the cells were successfully used in the study of PCV2 proliferation on this basis,which initially gave us a good effect,which provided technical basis for further research on antiviral proteins and the development of highly efficient PCV2 susceptible cell lines.