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目的构建小鼠Tim-3真核表达载体,并在黑色素瘤细胞系B16中表达小鼠TIM-3。方法以脾细胞RNA为模板,逆转录PCR扩增鼠Tim-3编码区基因,T-A克隆至真核表达载体pTARGET,构建重组质粒pTARGET-Tim-3,采用脂质体转染法转染黑色素瘤细胞系B16细胞,以逆转录PCR和Westernblot验证Tim-3在B16细胞中的表达。结果利用酶切和测序的方法,筛选、鉴定pTARGET-Tim-3真核表达载体,转染黑色素瘤细胞系B16细胞,经逆转录PCR和Westernblot证实TIM-3高效表达。结论成功构建小鼠Tim-3真核表达载体,Tim-3在转染的小鼠B16细胞系中高表达。
Objective To construct mouse Tim-3 eukaryotic expression vector and express mouse TIM-3 in melanoma cell line B16. Methods The spleen cell RNA was used as a template to amplify the gene encoding Tim-3 in rat by RT-PCR and cloned into eukaryotic expression vector pTARGET. The recombinant plasmid pTARGET-Tim-3 was constructed and transfected into melanoma by lipofection Cell line B16 cells, the expression of Tim-3 in B16 cells was verified by reverse transcription PCR and Western blot. Results The eukaryotic expression vector pTARGET-Tim-3 was screened and identified by restriction endonuclease digestion and sequencing. The transfected melanoma cell line B16 was confirmed by RT-PCR and Western blot. Conclusion The mouse Tim-3 eukaryotic expression vector was successfully constructed and Tim-3 was highly expressed in the transfected mouse B16 cell line.