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

作　　者：孟洁洁 宋月[1] 樊文杰[1] 杨乐 邢嘉友 王江[1] 褚贝贝[1] 杨国宇 王梦迪 MENG Jiejie;SONG Yue;FAN Wenjie;YANG Le;XING Jiayou;WANG Jiang;CHU Beibei;YANG Guoyu;WANG Mengdi(Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affiars,Henan Agricultural University,Zhengzhou 450046,China;College of Food and Bioengineering,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China)

机构地区：[1]河南农业大学,农业农村部动物生化与营养重点开放实验室,郑州450046 [2]河南牧业经济学院,食品与生物工程学院,郑州450046

出　　处：《中国畜牧兽医》2022年第6期2011-2021,共11页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金面上项目(32072858)。

摘　　要：【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【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

关 键 词：CRISPR/Cas9 TLR7基因 猪肾上皮细胞(PK15) 水疱性口炎病毒(VSV) 

分 类 号：S852.659.5[农业科学—基础兽医学]