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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 a recombinant eukaryotic expression vector containing the target gene to obtain L929 gene transfected cells stably expressing human Tim-3. Methods The gene of Tim-3 was cloned from human peripheral blood T lymphocytes by RT-PCR. The gene was inserted into eukaryotic expression vector pIRES2-EGFP by XhoI and Sa-lI, and then transfected into L929 After 72 h, G418 was selected for screening, and L929 cell line stably expressing Tim-3 protein was selected. RESULTS: The recombinant eukaryotic expression vector containing Tim-3 gene was constructed and transfected into L929 cells by lipofectamine 2000. Then RT-PCR and flow cytometry were used to detect the expression of human Tim-3 Protein L929 Transgenic Cells. Conclusion The recombinant eukaryotic expression vector containing human Tim-3 gene and the cell line stably expressing human Tim-3 protein were constructed, which laid the foundation for the further study on the function of this gene and the development of monoclonal antibodies.