Toll样受体7基因敲除对水疱性口炎病毒增殖的影响

Effect of Toll-like Receptor 7 Gene Knockout on Proliferation of Vesicular Stomatitis Virus

【目的】探究敲除Toll样受体7(Toll-like receptor 7,TLR7)基因对水疱性口炎病毒(Vesicular stomatitis virus,VSV)复制的影响。【方法】利用慢病毒介导的CRISPR/Cas9基因编辑技术构建TLR7基因敲除的稳定猪肾上皮细胞(porcine renal epithelial cells,PK15)系。通过构建pTLR7-sgRNA重组质粒并转染至HEK293T/17细胞,收获慢病毒并感染PK15细胞,经嘌呤霉素筛选后得到PK15-TLR7^(-/-)多克隆细胞,并在Western blotting鉴定后通过有限稀释法获得PK15-TLR7^(-/-)单克隆稳定细胞系。为验证敲除TLR7基因稳定细胞系是否构建成功,分别利用光学显微镜和细胞毒性检测(cell counting kit 8,CCK-8)观察并检测PK15、PK15-TLR7^(-/-)细胞的形态与活力;利用荧光倒置显微镜和流式细胞术观察VSV-GFP感染PK15、PK15-TLR7^(-/-)细胞后病毒增殖情况;利用Western blotting和实时荧光定量PCR分别检测VSV-GFP感染PK15、PK15-TLR7^(-/-)细胞后GFP蛋白和VSV-N基因mRNA水平的表达情况;利用病毒滴度检测VSV-GFP感染PK15、PK15-TLR7^(-/-)细胞后子代病毒产生情况。【结果】采用CRISPR/Cas9基因编辑技术构建的3种sgRNA均对TLR7进行了有效的编辑,其中sgRNA2的基因编辑效率最高,但敲除TLR7基因并不影响PK15细胞的形态及活力;当感染VSV-GFP后,通过荧光显微镜观察发现,荧光强度随着时间逐次增加,且PK15-TLR7^(-/-)细胞的GFP荧光强度强于PK15细胞。流式细胞术检测结果显示,相同时间点的PK15-TLR7^(-/-)细胞感染VSV-GFP的比例显著或极显著高于PK15细胞(P<0.05;P<0.01);实时荧光定量PCR结果表明,感染4~36 h VSV-N基因mRNA相对表达量随着时间逐渐增加,且PK15细胞中VSV-N基因的mRNA相对表达量显著或极显著低于PK15-TLR7^(-/-)细胞(P<0.05;P<0.01);Western blotting结果表明,PK15与PK15-TLR7^(-/-)细胞中的VSV-GFP GFP蛋白均从6 h开始表达,表达量随时间逐渐增多,且在PK15-TLR7^(-/-)细胞中VSV-GFP GFP蛋白含量比PK15细胞更高;感染VSV-GFP 6 h后子代病毒开始释放,此时PK15细胞中VSV-GFP子代病毒滴度低于PK15-TLR7^(-/-)细胞(P>0.05),但PK15细胞中VSV-GFP子代病毒滴度随着感染时间的增加显著或极显著低于PK15-TLR7^(-/-)细胞(P<0.05;P<0.01)。【结论】TLR7基因的敲除可以促进VSV在PK15细胞中的复制,初步验证了TLR7在天然免疫中的作用,为VSV等RNA病毒性疾病的防控提供新思路。

【Objective】The objective of this study was to investigate the effects of Toll-like receptor 7(TLR7)gene knockdown on the replication of Vesicular stomatitis virus(VSV).【Method】The stable porcine renal epithelial cells(PK15)with TLR7 gene knockout were constructed using lentivirus-mediated CRISPR/Cas9 gene editing technique.The recombinant plasmid pTLR7-sgRNA was constructed and transfected into HEK293T/17 cells.Lentivirus was harvested and infected with PK15 cells.PK15-TLR7^(-/-)polyclonal cells were obtained after screening by purinomycin.PK15-TLR7^(-/-)monoclonal stable cell lines were obtained by limited dilution method after Western blotting.To verify the successful construction of TLR7 gene stabilized cell lines,optical microscopy and cytotoxicity test(CCK-8)were used to observe and detect the differences in morphology and viability of PK15 and PK15-TLR7^(-/-)cells.The proliferation of VSV-GFP infected PK15 and PK15-TLR7^(-/-)cells was observed by inverted fluorescence microscope and flow cytometry.Western blotting and Real-time quantitative PCR were used to detect the expression of GFP protein and VSV-N gene mRNA in PK15 and PK15-TLR7^(-/-)cells infected with VSV-GFP.Virus titers were used to detect the progenitor virus production of PK15 and PK15-TLR7^(-/-)cells infected with VSV-GFP.【Result】Three sgRNAs constructed by CRISPR/Cas9 gene editing technology could effectively edit TLR7,and sgRNA2 had the highest editing efficiency.However,TLR7 knockout did not affect the morphology and viability of PK15 cells.When infected with VSV-GFP,the fluorescence intensity increased with time,and the GFP fluorescence intensity of PK15-TLR7^(-/-)cells was stronger than that of PK15 cells.Flow cytometry results showed that the proportion of PK15-TLR7^(-/-)cells infected with VSV-GFP was significantly or extremely significantly higher than that of PK15 cells at the same time(P<0.05;P<0.01).Real-time quantitative PCR results showed that the mRNA relative expression of VSV-N gene increased gradually with time from 4 to 36 h after infection,and the mRNA relative expression of VSV-N gene in PK15 cells was significantly or extremely significantly lower than that in PK15-TLR7^(-/-)cells(P<0.05;P<0.01).Western blotting results showed that VSV-GFP GFP was expressed in PK15 and PK15-TLR7^(-/-)cells at 6 h,and the expression level increased gradually with time,and the content of VSV-GFP GFP in PK15-TLR7^(-/-)cells was higher than that in PK15 cells.After 6 h of VSV-GFP infection,the progeny virus was released and the titer of VSV-GFP progeny virus in PK15 cells was lower than that in PK15-TLR7^(-/-)cells(P>0.05).The virus titer of VSV-GFP progeny in PK15 cells was significantly or extremely significantly lower than that in PK15-TLR7^(-/-)cells with increasing infection time(P<0.05;P<0.01).【Conclusion】TLR7 gene knockout could promote VSV replication in PK15 cells,which preliminarily verified the role of TLR7 in natural immunity,providing new ideas and strategies for the prevention and control of VSV and other RNA viral diseases.