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

摘要/Abstract

摘要： 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Western blot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PRV mRNA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10^6.8 TCID₅₀·0.1 mL^-1，而在PK-15-TBK1^-/-细胞中复制后TCID₅₀为10^8.5 TCID₅₀·0.1 mL^-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。

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.

中图分类号:

S852.659.1

刘晓贺, 巴根, 李坚, 韩莹倩, 张爽, 明胜利, 杜永坤, 褚贝贝, 杨国宇, 王江. 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[J]. 畜牧兽医学报, 2019, 50(6): 1239-1248.

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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