The Construction Of Pig Kidney Epithelial Cell Line With Type ? Interferon Receptor 1(IFNAR1) Knockout

Type ? interferon are cytokines that play a key role in the innate immune system.Interferon-?/? receptor(IFNAR)binds to type 1 interferon and activates downstream signal transduction pathways,such as JAK-STAT,to induce the transcription of interferon stimulated gene(ISG).Low-affinity subunit type ? interferon receptor 1(IFNAR1)is an essential component in ensuring complete IFNAR activation.In this study,the porcine kidney epithelial PK15 cell line with IFNAR1 stably knockout was constructed by the lentiviral-mediated CRISPR/Cas9 genome editing technique.First,the single-stranded guide RNA(sgRNA)was designed and the recombinant plasmid pIFNAR1-sgRNA was constructed;then pIFNAR1-sgRNA was co-transferred with pMD2.G and psPAX2(lentiviral packaging plasmids)into human embryonic kidney cells(Human embryonic kidney 293T/17,HEK293T/17),and lentiviral solution was collected;PK15 cells were infected with this lentiviral solution and the polyclonal IFANR1 knockout cell line was obtained by puromycin selection;finally monoclonal PK15-IFNAR1-/-cell line was obtained by limiting dilution method.The absorbance between PK15-WT and PK15-IFNAR1-/-cells treated with CCK-8 was detected by microplate reader to determine whether IFNAR1 gene knockout affected cell proliferation.Fluorescence microscopy and flow cytometry were used to detect the fluorescent intensity in PK15-WT and PK15-IFNAR1-/-cells infected with PRV-GFP.Western blot was used to detect PRV-QXX gE protein expression between PK15-WT and PK15-IFNAR1-/-cells.Viral titer detection was used to determine PRV-QXX infection in PK15-WT and PK15-1FNAR1-/-cells.Real-time quantitative PCR was used to detect the transcription of PRV-QXX TK mRNA and interferon-stimulated gene 15(ISG 15)between PK15-WT and PK15-IFNAR1-/-cells.The pIFNAR1-sgRNA recombinant plasmid was successfully constructed.The results of T7 endonuclease I showed that the plasmid exhibited high genome editing efficiency in PK15 cells.The monoclonal PK15-IFNAR1-/-cell line derived from polyclonal cell lines was verified by DNA sequencing.The CCK-8 proliferation assay showed IFNAR1 knockout did not affect cell proliferation.Fluorescence microscopy and flow cytometry results showed that IFNAR1 knockout promoted PRV-GFP replication.Western blot assay showed that IFNAR1 knockout increased the expression of PRV-QXX gE protein.Viral titer assay results showed that IFNAR1 knockout significantly enhanced viral replication.The results of real-time quantitative PCR showed that the transcription level of ISG15 gene in PK15-WT cells was significantly increased with the prolongation of PRV-QXX infection,but there was no significant change in PK15-IFNAR1-/-cells.Viral TK gene transcription levels were significantly up-regulated in PK15-IFNAR1-/-compared to PK15-WT cells.In summary,the results showed that the PK15-IFNAR1-/-monoclonal cell line was successfully constructed,and knockout of the IFNAR1 gene promoted PRV replication.This study provides a cellular tool for further study of IFNAR1 gene function.