人Tim-3基因克隆及其转基因细胞的构建

Cloning of Human Tim-3 Gene and Construction of Its Transgenic Cells

目的克隆人Tim-3基因,并构建含有该目的基因的重组真核表达载体,获得稳定表达人Tim-3分子的L929基因转染细胞。方法采用RT-PCR方法从人外周血T淋巴细胞中克隆出Tim-3基因,通过双酶切(XhoI,Sa-lI)装入真核表达载体pIRES2-EGFP中,脂质体法转染L929细胞,72 h后加入G418进行筛选,挑选出能稳定表达Tim-3蛋白的L929细胞株。结果构建了用于表达的含Tim-3基因的重组真核表达载体,经脂质体转染L929细胞,继而经RT-PCR和流式细胞术表型检测,筛选出稳定表达人Tim-3蛋白的L929转基因细胞。结论构建了含人Tim-3基因重组真核表达载体和稳定表达人Tim-3蛋白的细胞株,为该基因功能的后续研究和单克隆抗体的研制奠定了基础。

Objective To clone human Tim-3 gene and construct the recombinant eukaryotic expression vector carriying the target gene which can be expressed stably in mammal cell line L929.Methods Human Tim-3 gene was amplified by RT-PCR using the mRNA of T cells from human peripheral blood as template,and then confirmed by DNA-sequence analysis.Digested with the restriction endonucleases XhoI and SalI,the human Tim-3 gene was inserted into eukaryotic expression vector pIRES2-EGFP.The recombinant plasmid was transfected into mammal cell line L929 using LipfectAMINE.After 72 hours,L929 cell line stably expressing human Tim-3 protein was selected in the presence of G418.Result The full-length of human Tim-3 gene was cloned and the recombinant eukaryotic expression vector was constructed,the L929 cell line expressing human Tim-3 was successfully transfected and selected.Conclusion The results of RT-PCR and flow cytometry indicates that L929 transgenic cells could stably express human Tim-3 protein on the membrane of cells.