免疫检查点TIGIT慢病毒表达载体的构建和稳定表达TIGIT细胞系的建立  

Construction of immune checkpoint TIGIT lentivirus expression vector and establishment of cell line stably expressing TIGIT

作　　者：穆业腾 郭冲 胡楠楠 杨馥旭 薛晗 范宇鑫 郭峰霖 关新刚 MU Yeteng;GUO Chong;HU Nannan;YANG Fuxu;XUE Han;FAN Yuxin;GUO Fenglin;GUAN Xingang(Key Laboratory of Pharmaceutics and Bioengineering,School of Medical Technology,Beihua University,Jilin 132013,China;Department of Basic Medicine,School of Medical Science,Taizhou University,Taizhou 318000,China)

机构地区：[1]北华大学医学技术学院医药生物工程重点实验室,吉林吉林132013 [2]台州学院医学院基础医学系,浙江台州318000

出　　处：《吉林大学学报（医学版）》2022年第5期1341-1347,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目(20180101213JC);台州学院高层次人才科研启动项目(T20220101026)。

摘　　要：目的:构建T细胞免疫球蛋白和免疫受体酪氨酸抑制基序结构域-绿色荧光蛋白(TIGITGFP)慢病毒表达载体,建立稳定表达TIGIT-GFP的细胞系,探讨TIGIT-GFP融合蛋白在稳定表达细胞系中的表达情况。方法:将TIGIT质粒和慢病毒载体pLenti-GFP分别采用限制性内切酶EcoRⅠ和NotⅠ双酶切,凝胶回收后连接构建重组质粒pLenti-TIGIT-GFP。将测序正确的重组质粒转染人胚胎肾HEK293T细胞作为实验组,转染TIGIT质粒的HEK293T细胞作为对照组,转染48 h后荧光显微镜下观察细胞膜上GFP表达情况。将实验组细胞继续培养,采用嘌呤霉素筛选2周后,挑取单克隆扩大培养,荧光显微镜观察细胞膜上GFP表达情况,Western blotting法检测在转染的人胚胎肾HEK293T细胞中TIGIT-GFP蛋白表达情况。结果:酶切鉴定,TIGIT基因成功插入pLenti-GFP表达载体,DNA测序未检测到突变发生。实验组细胞膜上检测到GFP表达。Western blotting法检测,实验组细胞裂解液中检测到TIGIT-GFP特异性条带。结论:成功构建pLenti-TIGIT-GFP慢病毒表达载体,建立稳定表达TIGIT-GFP融合蛋白的细胞系,TIGIT-GFP融合蛋白主要在稳定细胞系的细胞膜上表达。Objective:To construct the lentiviral expression vector of T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain-green fluorescent protein(TIGIT-GFP) and establish a cell line stably expressing TIGIT-GFP,and to explore the expression of TIGIT-GFP fusion protein in stable expression cell line. Methods:The TIGIT plasmid and lentiviral vector plenti-GFP were doubly digested by endonucleases EcoR Ⅰ and Not Ⅰ. The inserted gene and vector in agarose gel were recovered and ligated to construct the recombinant plasmid pLenti-TIGIT-GFP. The human embryonic kidney HEK293T cells transfected with sequenced recombinant plasmid were used as experimental group,and the HEK293T cells transfected with pCMV6-TIGIT were used as control group. After 48 h of transfection,the expression of GFP in the cell membrane was detected under fluorescence microscope. The cells in experimental group were cultured and screened with puromycin for two weeks;the screened cells were selected and expanded,and the expression of TIGIT-GFP protein in cell membrance was examined by fluorescence microscope. Western blotting method was used to determine the TIGIT-GFP protein expression in monoclonal cells. Results:The enzyme digestion results showed that TIGIT gene was inserted into the expression vector pLenti-GFP,and the DNA sequencing showed no mutation. The GFP in the cell membrane in experimental group was found. The Western blotting results showed a specific band of TIGIT-GFP in cell lysis buffer in experimental group. Conclusion:The lentiviral expression vector pLentiTIGIT-GFP is successfully established,the cell line stably expression TIGIT-GFP fusion protein is constructed,and the TIGIT-GFP protein is mainly expressed in the cell membrane of the stable cell line.

关 键 词：T细胞免疫球蛋白和免疫受体酪氨酸抑制基序结构域 绿色荧光蛋白 慢病毒表达载体 稳定转染 单克隆细胞 

分 类 号：R392.2[医药卫生—免疫学]