中文摘要：

目的克隆人Tim-3基因5’端非编码区处具有启动子活性的片段，构建其真核报告质粒，为进一步研究Tim-3表达调控奠定基础。方法以人基因组DNA为模板，针对人Tim-3启动子5’端非编码区处包括翻译起始点ATG和转录起始点TSS在内的-898～＋242片段。设计特异性引物，PcR扩增该片段，命名为Tim-3P2，经双酶切后克隆入pGL3-Basic载体SaeⅠ、XhoⅠ位点，构建pGL3-Tim-3P2荧光素酶报告质粒，与内参照pRL-TK用脂质体法瞬时共转染COS-7、RAW264．7和B16细胞系。通过双荧光素酶活性检测确定其启动子活性。结果rGL3-Tim-3P2经PCR、双酶切及DNA测序分析鉴定正确无误；pGL3-Tim-3P2转染RAW264．7和B16细胞（两种细胞均可表达内源性Tim-3）24h和48h，双荧光素酶活性检测显示其启动子相对活性约为pGL3-Basic空载体的2倍；而pGL3-Tim-3P2转染不表达内源性Tim-3的COS-7细胞后检测不到荧光素酶活性。结论成功克隆并构建含有人Tma-3启动子的真核报告质粒。该载体具有特异性Tim-3启动子活性，为进一步研究Tim-3表达调控奠定了基础。

英文摘要：

Objective To construct a lucifemse reporter plasmid containing human T-cell immunnglobulin- 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 perfomed in different cell lines, pRL-TK was used to normatize the transfection etliciency. 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 maerophage 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 suecessfully constructed which could selectively express in Tim-3 positive cells.