基于慢病毒包装和CRISPR技术建立病毒感染关键基因CLTC敲除细胞系  

Construction of Key Gene CLTC Knockout Cell Line Based on Lentivirus Packaging and CRISPR Technology

作　　者：刘铁柱[1] 黄涛[1] 李阿茜 李川[1] 梁米芳[1] 王世文[1] LIU Tiezhu;HUANG Tao;LI Aqian;LI Chuan;LIANG Mifang;WANG Shiwen(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention.Key laboratory for Medical VirologyandViral Disease,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,医学病毒和病毒病重点实验室,北京102206

出　　处：《病毒学报》2023年第3期800-804,共5页Chinese Journal of Virology

基　　金：国家重点研发计划任务(项目号:2022YFC2303402),题目:病原宿主互作机制研究及广谱新靶点发现。

摘　　要：网格蛋白是病毒进入内体系统时的关键蛋白,同时还可能参与病毒的胞内转运,目前该蛋白的具体作用范围报道还不一致。为了构建网格蛋白重链基因敲除细胞系,以研究其在病毒感染过程中的功能,本研究基于CRISPR/Cas9系统,首先通过包装慢病毒建立网格蛋白重链基因特异性打靶载体,使用嘌呤霉素对慢病毒感染的Huh7细胞进行阳性筛选,利用96孔板梯度稀释法获得单克隆细胞,提取细胞基因组DNA,PCR扩增后进行测序验证,同时提取细胞蛋白,利用Western Blot实验进行蛋白敲除验证,最后进行细胞增殖活性检测和脱靶效应评估。最终本研究获得了一株网格蛋白重链基因敲除Huh7细胞系;通过脱靶效应评估,显示最可能的10个脱靶位点均不存在脱靶现象,且该基因敲除不影响细胞增殖活性。本研究成功构建了一株网格蛋白重链基因敲除Huh7细胞系,为研究网格蛋白在病毒进入宿主细胞时的功能建立了细胞模型。Clathrin is a key protein for the entry of the virus into the endosomal system,and may also be involved in the intracellular transport of virus.However,the specific range of functions of clathrin have not been reported in agreement.We aimed to construct clathrin heavy chain knockout cell lines to investigate their function during viral infection.Based on CRISPR/Cas9 system,we constructed a lentivirus vector targeting human CLTC gene.Then,we screened Huh7 cells positive for lentivirus infection with puromycin,and we obtained single cells by gradient dilution in 96-well plates.Genomic DNA was extracted and amplified by polymerase chain reaction for sequencing verification.Simultaneously,proteins were extracted and verified by western blotting analysis.Cell activity was detected and off-target effects evaluated.Finally,a CLTC-knockout Huh7 cell line was obtained.There were no off-target effects in the top 10 off-target sites.CLTC gene knockout showed no effect on cell proliferation.We constructed a CLTC-knockout Huh7 cell line,which established a cell model for functional study of clathrin protein and mechanism of virus entry into host cells.

关 键 词：CRISPR/Cas9 CLTC 基因敲除 

分 类 号：R373.1[医药卫生—病原生物学]