A Preliminary Study Of Immune Effect In Cells Infected By Porcine Pseudorabies Virus

Biological safety is fundamental to pseudorabies virus vaccine research and development.At the cellular level,analyses on the innate immune response and apoptosis are pivotal in assessment of the safety of TK,gE single and double knockout virus vaccines.And the construction of certain gene knockout cell lines can provide a intergral cell model for the research of antiviral innate immune response.To successfully prepared Tlr3-/-、Tlr7-/-、Stat6-/-nd Rigl-/-knockout cell lines,we take a full advantage of the CRISPR/Cas9 system.And the CRISPR/Cas9 plasmid vector,PX459(containing the Cas9 and puromycin hormone resistance genes),is transfected successfully into wild type pk-15 cells by using Lipofectamine 3000 and then selected puromycin-resistant pk-15 cells with 2 μg/mL puromycin solution.When the confluence of selected cells reaches 80%,their genomic DNAs were isolated for PCR sequencing to confirm whether the selected genes have been edited.Then the hybrid cells were diluted,and the monoclonal cell lines containing the modifications of corresponding genes including Tlr3、Tlr7、Stat6 and Rig1,were isolated and identified by T-A cloning and sequencing.These monoclonal cells were further verified by protein expression analyses using Western blotting.Finally,four gene-edited cell lines were successfully obtained and named as pk-15-Rig1-/-、pk-15-Tlr3-/-、pk-15-Tlr7-/-、pk-15-Stat6-/-.In the study,wild type PRV,PRV-TK-,PRV-gE-and PRV-TK-/gE-virus strains were used to infect pk-15-Tlr3-/-cells,pk-15-Tlr7-/-cells and pk-15-Stat6-/-cells in order to test the gene knockout effects and the interations among the genes.By analyzing the changes of IFN-β,IFIT1,CXCL10,IL-6 and Bcl-2 in the gene deletion cells of Tlr3-/-Tlr7-/-and Stat6-/-,following the treatments of wild type PRV,PRV-TK-,PRV-gE-and PRV-TK-/gE-virus strains.The data are summarized in the following:1)Both gE and TK genes in the presence and absence of infected pseudorabies virus strains(cells)are able to activate antiviral innate immune pathway mediated by Tlr3 and Tlr7,however,the deletion of gE and TK gene could down regulate the expression of type I interferon;2)gE and TK have an antagonistic relationship with Stat6 in the antiviral innate immune response;3)gE and TK genes may have some mechanism in the inhibition of cell self-protection.These studies provide the essential evidence for the mechanisms involved in the PRV infection and for the future clinical application of these viral strains as the vaccines for preventing and treating PRV infection.