PK-15细胞敲除TANK结合激酶1基因促进猪伪狂犬病病毒复制的研究

doi:10.11843/j.issn.0366-6964.2019.06.014

Abstract

Abstract: TANK-binding kinase 1(TBK1) is a key enzyme responsible for IRF3, IRF7 phos-phorylation and type Ⅰ interferons expression during viral infection. Furthermore, TBK1 is an important element in antiviral natural and acquired immune response. To study the effect of TBK1 gene knockout on porcine pseudorabies virus (PRV) replication, in this study, TBK1 knockout PK-15 cell line was constructed by CRISPR/Cas9 technology. Next, the cell viability of PK-15-TBK1^-/- cells was monitored by CCK-8 assay. Then, the following indexes were used to comprehensively evaluate the effect of TBK1 gene knockout on PRV replication:fluorescence intensity of PRV-GFP was assayed by flow cytometry; mRNA levels of PRV-gB, PRV-gE, PRV-TK, IL-1β, IFN-β and ISG15 were measured by RT-qPCR; protein expression levels of PRV-gB and PRV-gE were evaluated by Western Blot; infectivity of progeny virus was determined by titer determination. Results were as follows:Firstly, T7E1 assay results showed that target bands were identified from 3 sgRNA target sites in exon 2 regions of TBK1 gene. TBK1-sgRNA1 cells with highest editing efficiency were selected with limiting dilution method for monoclonal cultivation by inoculating into a 96-pore plate. Then, No.4 cell strain from 6 independent TBK1 stable knockout monoclonal cells was chosen for CCK-8 assay. The results showed that knockout of TBK1 gene had no effect on cell viability. In addition, flow cytometry results showed that positive cells infected with PRV-GFP account for 56.89% of the total PK-15 cells, and those were 77.95% of the total PK-15-TBK1^-/- cells, indicating that PK-15-TBK1^-/- cells could enhance PRV replication. Moreover, RT-qPCR and WB results showed that PK-15-TBK1^-/- cells could up-regulate PRV mRNA transcription and protein translation. Titer determination showed that the TCID₅₀ of new progeny virions in PK-15 cells and PK-15-TBK1^-/- cells were 10^6.8 TCID₅₀·0.1 mL^-1 and 10^8.5 TCID₅₀·0.1 mL^-1, respectively. Besides, RT-qPCR results showed that up-regulation transcription of IL-1β, IFN-β and ISG15 induced by PRV infection were resisted in PK-15-TBK1^-/- cells. In conclusion, the above results indicate that knockout of TBK1 gene promotes PRV replication in PK-15 cells, which might be linked to the inhibition of transcription of IL-1β, IFN-β and ISG15.

CLC Number:

S852.659.1

LIU Xiaohe, BA Gen, LI Jian, HAN Yingqian, ZHANG Shuang, MING Shengli, DU Yongkun, CHU Beibei, YANG Guoyu, WANG Jiang. Genetic Knockout of TANK-binding Kinase 1 Gene in PK-15 Cells Promotes Pseudorabies Virus Replication[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1239-1248.

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Genetic Knockout of TANK-binding Kinase 1 Gene in PK-15 Cells Promotes Pseudorabies Virus Replication

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