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

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

TANK结合激酶1(TBK1)是病毒感染时IRF3、IRF7磷酸化及Ⅰ型干扰素表达的关键酶,在抗病毒天然免疫应答和获得性免疫应答中发挥重要作用。为研究TBK1基因敲除对猪伪狂犬病病毒(PRV)复制的影响,首先利用CRISPR/Cas9技术构建猪TBK1基因稳定敲除PK-15细胞系,并利用CCK-8检测敲除TBK1对PK-15细胞活力的影响。然后利用流式细胞术检测PRV-GFP荧光强度,用RT-qPCR检测PRV-gB、PRV-gE、PRV-TK、IL-1β、IFN-β和ISG15的转录水平,用Westernblot检测PRV-gB和PRV-gE蛋白表达水平,以及通过滴度测定检测子代病毒的感染力,综合评价TBK1基因稳定敲除PK-15细胞对PRV病毒复制的影响。结果显示:T7E1检测结果显示TBK1基因外显子2区的3个sgRNA靶位点均切出了目的条带,选取编辑效率最高的TBK1-sgRNA1细胞系进行单克隆化培养,获取6株稳定敲除TBK1基因的单克隆细胞,随机选取4号细胞株进行CCK-8检测,结果显示敲除TBK1对PK-15细胞活力无影响。流式检测结果显示PRV-GFP感染阳性细胞占总PK-15细胞的56.89%,PRV-GFP感染阳性细胞占总PK-15-TBK1-/-细胞的77.95%,表明PK-15-TBK1-/-细胞可以促进PRV-GFP复制。RT-qPCR及WB检测显示该细胞系可以促进PRVmRNA转录和蛋白表达。滴度测定结果显示,PRV-QXX在PK-15细胞复制后TCID50为10^6.8TCID50·0.1mL^-1,而在PK-15-TBK1-/-细胞中复制后TCID50为10^8.5TCID50·0.1mL^-1。另外,RT-qPCR结果显示该细胞系可以抑制PRV感染引起的IL-1β、IFN-β和ISG15转录上调。以上结果表明TBK1基因敲除PK-15细胞系促进PRV复制,可能与IL-1β、IFN-β和ISG15转录水平抑制有关。

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 TBK 1 gene knockout on porcine pseudorabies virus (PRV) replication,in this study, TBK 1 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 TBK 1 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 TBK 1 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 TBK 1 stable knockout monoclonal cells was chosen for CCK-8 assay. The results showed that knockout of TBK 1 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 TCID 50 of new progeny virions in PK-15 cells and PK-15-TBK1 -/- cells were 10^6.8 TCID 50 ·0.1 mL^-1 and 10^8.5 TCID 50 ·0.1 mL^-1 ,respectively. Besides,RT-qPCR results showed that up-regulation transcription of IL-1β, IFN-β and ISG 15 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 ISG 15.