Establishment Of A Porcine PARP11 Knockout PK-15 Cell Line Susceptible To Porcine Virus

Swine virus has seriously affected the healthy development of pig industry and caused huge economic losses.Using CRISPR / Cas9 technology to edit the genes related to viral replication and proliferation in the host,then to study the pathogenesis of the host,and to explore the application of viral therapy is an important way to study viral therapy.ADP ribosyltransferase poly(ADP ribosyltransferase)polymerase(PARP)family plays an important role in the life activities of the body.It can affect a variety of physiological processes of cells,such as DNA repair and apoptosis,telomere homeostasis,mitotic separation,cell cycle regulation,cell differentiation and epigenetic modification.PARP11 is a member of PARP protein family.At present,there are few studies on PARP11 gene,and its specific biological function is not clear.Recent studies have shown that PARP11 through mono ADP-ribosylation of ubiquitin E3 ligaseβ-Transduction repeat protein(β-Trcp)plays an important role in the innate immune response.In this study,we used CRISPR /Cas9 as the basis of efficient site-specific gene editing pig platform to explore the effect of porcine PARP11 gene on four common porcine viruses infection and the related molecular mechanism.The results are as follows(1)Two pairs of sg RNAs(sg RNA1 and sg RNA2)targeting the third exon of porcine PARP11 gene were designed and synthesized.The editing efficiency of the designed sg RNAs was identified by sequencing and nested peak analysis.The results showed that the editing efficiency of sg RNA1 and sg RNA2 was 11.3% and 7.9%,respectively;(2)Based on the selected sg RNA1 which can efficiently edit porcine PARP11 gene,PX330 plasmid was introduced into PK-15 cells by electroporation.Then,different genotypes of PK-15 cell clones with PARP11 gene knockout were obtained by limiting dilution planing strategy.Western blot results further showed that,compared with the control group,the expression of PARP11 gene in PK-15 cells was significantly increased,The protein translation level of these PARP11 knockout PK-15 cells decreased;(3)In order to further explore the effect of knockout of porcine PARP11 gene on the susceptibility of porcine viruses,in the next experiment,the infection of PEDV,PCV2,PRV,CSFV in PK-15 cells with knockout of porcine PARP11 gene were studied.The results showed that PK-15 cells with knockout of porcine PARP11 gene significantly promoted the replication and proliferation of CSFV and PEDV;However,the results of PCV2 and PRV infection showed that PARP11 knockout PK-15 cells significantly inhibited the replication and proliferation of PCV2 and PRV;(4)Finally,this study explored the potential mechanism of PARP11 gene knockout affecting the host cells infected by the virus from the aspect of PARP11 regulating IFN-I signaling.The results showed that the expression level of IFN-I related factors in PK-15 cells with knockout of PARP11 gene was significantly decreased when infected with CSFV;When PCV2 was infected,the expression level of IFN-I related factors increased significantly.In this study,different genotypes of porcine PARP11 knockout PK-15 cell clones were successfully obtained by CRISPR / cas9 mediated site directed knockout strategy.Based on this cell clone,we further studied the effect of PARP11 gene on the susceptibility of porcine virus,and preliminarily explored the potential mechanism of PARP11 gene knockout affecting the host cells infected by the virus from the aspect of PARP11 regulating IFN-I signal.In conclusion,this study provides an effective target for immune response and antiviral research of pigs,and provides a new therapeutic strategy for the prevention and treatment of porcine viruses.