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目的：为了构建可在真核细胞中高效表达人Tim-3（hTim-3）的真核表达质粒,以便用于hTim-3 肿瘤免疫治疗研究。方法：取健康人外周血的单个核细胞，用高保真聚合酶扩增hTim-3 基因，先进行hTim-3 基因的亚克隆，用BglⅡ和SalⅠ限制性内切酶切下带有酶切位点的hTim-3 基因，最终构建hTim-3 基因的真核表达质粒pEGFP-N1-hTim-3。用脂质体方法转染质粒至肝癌细胞 SMMC7721 和巨噬细胞U937 中。48h 后荧光显微镜观察转染细胞绿色荧光表达情况，初步判断转染效率。结果：酶切和测序鉴定证实目的基因hTim-3 正确插入到真核表达载体pEGFP-N1 中，转染肝癌细胞SMMC7721 和巨噬细胞U937 后，在荧光显微镜下观察到绿色荧光蛋白的表达。结论：成功构建了可在真核细胞中高效表达hTim-3 基因的真核表达质粒pEGFP-N1-hTim-3，为进一步研究hTim-3 的肿瘤免疫治疗奠定了基础。

英文摘要:

Objective: To construct eukaryotic expression plasmids which can efficiently expressHuman TIM-3 Gene in eukaryotic cells for the research of tumor immunology. Methods: The hTim-3 cDNA was particularly amplified with primers from the total RNA of the PBMC of the health person and then cloned to the vector pGEM-T. The pGEM-T-hTim-3 recombinant plasmid was purified and digested with BglⅡand SalⅠ, then the hTim-3 was inserted into the BglⅡand SalⅠsites of the eukaryotic expression plasmid pEGFP-N1. Use the method of lipofectine to transfect the plasmid into the SMMC7721 cell and macrophage U937. Observe the expression of GFP under the fluorescence microscope 48 hours later to detect the hTim-3 expression. Results: Digestion and sequencing demonstrate that pEGFP-N1-hTim-3 recombinant plasmid was successfully constructed. The expression of GFP in pEGFP-N1-hTim-3 transfected hepatocarcinoma cell SMMC7721 and macrophage U937 was observed and located at the membrane of the cell. It was consistent with thta the hTim-3 was the type I membrane protein and demonstrated that the fusion gene could be effectively translated. Conclusion: We have successfully constructed pEGFP-N1-hTim-3 eukaryotic expression plasmid. The newly constructed recombinant vector is useful for follow- up identification of the function of hTim-3 protein in different cells, and it lays the foundation for the follow-up tumor immunology research.

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