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

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（TIGIT-GFP）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EcoR Ⅰ和Not Ⅰ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。

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

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 pLenti-TIGIT-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.