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

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.