Construction Of TANK-binding Kinase 1 Gene Knockout PK15 Cell Line

Natural immunity is the first line of defense against the invasion of microorganisms.The TANK-binding kinase 1(TBK1)as a key enzyme is responsible for IRF3,IRF7 phosphorylation and type I interferons expression during viral infection.Furthermore,TBK1 is an important element in antiviral natural and acquired immune response.To study what role does TBK1 gene play of in the process of pseudorabies virus(PRV)replication,in this study,TBK1 gene knockout PK15 cell line was constructed by lentivirus-mediated CRISPR/Cas9 technology.Firstly,we design the single-stranded guide RNA(sgRNA)and connect to the vector plasmid LentiCRISPR v2,then lentivirus was obtained by transfection,and then,we used lentiviruses to infect cells and obtained polyclonal cell lines by puromycin pressure screening,finally,the monoclonal cell line of TBK1 gene knockout(PK15-TBK1-/-)was obtained by finite dilution method.Next,the cell viability ofPK15-TBK1-/-cells was monitored by CCK8 assay.Then,the following indexes were used to comprehensively evaluate the effect of TBK1 knockout on PRV replication:fluorescence intensity of PRV-GFP assayed by flow cytometry;mRNA expression levels of PRV gB,PRV gE,PRV TK,IL-1?,IFN-?and ISG15 measured by RT-qPCR;protein expression levels of PRV gB and PRV gE evaluated by Western Blot;infectivity of progeny virus determined by titer determination.The recombinant plasmid was successfully constructed by DNA sequencing and sequence alignment.T7E1 assay results showed that target bands were identified from 3 sgRNA target sites in exon 2 regions of TBK1.TBK1-sgRNA1 cells with highest editing efficiency were selected with limiting dilution method for monoclonal cultivation by inoculating into a 96-pore plate.Then,Number 4 cell strain fi-om 6 independent TBK1 stable knockout monoclonal cells was chosen for CCK-8 assay.The results showed that knockout of TBK1 had no effect on cell viability.In addition,flow cytometry results showed that positive cells infected with PRV-GFP was 56.89%of the total PK15 cells,and those was 77.95%of the total PK15-TBK1-/-cells indicating that PK15-TBK1-/-cells could enhance PRV replication.Moreover,RT-qPCR and WB results showed that PK15-TBK1-/-cells could up-regulate PRV mRNA transcription and protein translation.Titer determination showed that the TCID50 of new progeny virions in PK15 cells and PK15-TBK1-/-cells were 106 8 TCID50·0.1 mL-1 and 108.5 TCID50·0.1 mL-1,respectively.Besides,RT-qPCR results showed that up-regulation expression of IL-1?,IFN-? and ISG15 induced by PRV infection was resisted in PK15-TBK1-/-cells.In conclusion,the above results indicate that the cell line of TBK1 gene knockout was successfully constructed,and the cell line has a good availability,which in the study of the mechanism of PRV infection.