干扰素诱导跨膜蛋白3基因敲除Caco2细胞株的构建及其对水泡性口炎病毒复制的影响  

Establishment of IFITM3 gene knockout Caco2 cellstrain and effect on replication of VSV

作　　者：庞照霞 郝鹏飞 屈巧巧 李乐天 李昌 金宁一 PANG Zhaoxia;HAO Pengfei;QU Qiaoqiao;LI Letian;LI Chang;JIN Ningyi(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China)

机构地区：[1]西北农林科技大学动物医学学院,陕西杨凌712100 [2]中国农业科学院长春兽医研究所,吉林长春130122

出　　处：《中国兽医学报》2023年第3期476-482,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2021YFD1801103-6)。

摘　　要：通过CRISPR/Cas9系统,旨在构建干扰素诱导跨膜蛋白3(IFITM3)基因敲除的人结直肠腺癌细胞Caco2,并探究IFITM3基因敲除对水泡性口炎病毒(VSV)复制的影响。针对人IFITM3基因靶向设计sgRNA,合成后连接至pLentiv2载体中获得重组质粒,经测序后将正确连接sgRNA的重组质粒及辅助质粒psPAX2、pMD2.G共转染293T细胞进行慢病毒包装并感染Caco2细胞,然后进行嘌呤霉素加压筛选,利用有限稀释法获取IFITM3基因敲除单克隆细胞。利用DNA测序和Western blot对所获得单细胞克隆进行鉴定,鉴定正确的细胞命名为Caco2-△IFITM3。用表达绿色荧光蛋白的VSV(VSV-EGFP)感染Caco2-△IFITM3细胞,通过荧光显微镜观察IFITM3敲除对VSV复制的影响。基因组DNA测序结果表明,在IFITM3基因开放阅读框产生了碱基缺失,细胞中IFITM3蛋白表达消失,但细胞活性未受明显影响。病毒感染试验可观察到Caco2-△IFITM3细胞感染比例显著高于Caco2细胞,表明敲除IFITM3使其抗病毒功能减弱。本研究通过CRISPR/Cas9技术成功构建了基于肠道细胞敲除IFITM3基因的Caco2单细胞克隆,验证了IFITM3敲除对细胞抗VSV感染的影响,为进一步深入研究IFITM3与肠道病毒的互作及其功能奠定基础。The aim of this study was to construct IFITM3 gene knockout colorectal adenocarcinoma(Caco2)stable cell strain by CRISPR/Cas9 system and to investigate the effect of the IFITM3 knockout on the replication of vesicular stomatitis virus(VSV).Guide RNAs targeting the human IFITM3 gene were cloned into the pLentiv2 vector to obtain a recombinant plasmid.After sequencing,the recombinant plasmid and helper plasmids psPAX2 and pMD2.G were cotransfected into 293T cells to assemble lentivirus.Then,Caco2 cells were infected by the lentivirus and monoclonal Caco2 cells were attained using dilution method.Eventually,monoclonal cells were identified by DNA sequencing and Western blot,and correct monoclonal cells were named Caco2-△IFITM3.Caco2-△IFITM3 cells were infected with vesicular stomatitis virus expressing green fluorescent protein(VSV-EGFP)and the effect of IFITM3 knockout on VSV-EGFP replication was determined by fluorescence microscopy.The results indicated that there were bases deletions in the open reading frame of the IFITM3 gene and the expression of IFITM3 protein disappeared in Caco2-△IFITM3cells and a significantly higher proportion of VSV infection in Caco2-△IFITM3 cells was observed.In this study,we successfully constructed IFITM3 knockout Caco2 cell strain through CRISPR/Cas9 technology and verified the effect of IFITM3 knockout on cellular resistance to VSV infection,which provided cellular models to further study the interaction between IFITM3 and enterovirus.

关 键 词：干扰素诱导跨膜蛋白3(IFITM3) CRISPR/Cas9 Caco2细胞 水泡性口炎病毒(VSV) 

分 类 号：Q78[生物学—分子生物学] S852.65[农业科学—基础兽医学]