Construction of the luciferase reporter plasmid containing human Tim-3 promoter

Objective To construct a luciferase reporter plasmid containing human T-cell immunoglobulin-and mucin-domain-containing molecule-3(Tim-3) gene promoter.Methods The human Tim-3P2 was amplified by PCR using human genomic DNA as a template and was directionally cloned into pGL3-basic multiple cloning sites to construct the luciferase reporter plasmid pGL3-Tim-3P2.To detect the transcriptional activity of human Tim-3P2 in the plasmid,transient transfection was performed in different cell lines.pRL-TK was used to normalize the transfection efficiency.Results DNA sequencing verified the successful construction of the plasmid pGL3-Tim-3P2. The results of transient transfection showed that the recombinant plasmid could be lowly expressed in the murine macrophage cell line RAW264.7 and murine melanoma cell line B16, both of which can endogenously express Tim-3,but not in the African green monkey kidney cell line COS-7 in which no endogenous Tim-3 could be detected.Conclusion The recombinant plasmid containing human Tim-3P2 has been successfully constructed which could selectively express in Tim-3 positive cells.