论文部分内容阅读
目的:构建人TIM-3真核表达载体,在真核细胞中表达人TIM-3,建立稳定转染细胞株。方法:设计特异性引物,利用PCR技术扩增出TIM-3全编码区基因片段,插入到真核表达载体pIRES2EGFP中,重组质粒pIRES2EGFP-hTIM-3用脂质体转染法转染哺乳动物细胞,以流式细胞术(FCM)和Western blot分析蛋白质的的表达,借助FCM和选择性培养基筛选建立稳定转染细胞株。结果:成功构建了pIRES2EGFP-hTIM-3真核表达载体,转染哺乳动物细胞COS-7和CHO后,FCM和Western blot分析证实TIM-3高效表达于细胞膜表面,建立了稳定转染的CHO细胞株。结论:成功构建了人TIM-3真核表达载体,TIM-3高效表达在转染的哺乳动物细胞表面,并建立了稳定转染的细胞株。为进一步研究TIM-3及其配体的生物学功能以及制备和筛选抗人TIM-3mAb提供了条件。
Objective: To construct human TIM-3 eukaryotic expression vector, express human TIM-3 in eukaryotic cells and establish stable transfected cell lines. Methods: Specific primers were designed. The full-length TIM-3 gene fragment was amplified by PCR and inserted into the eukaryotic expression vector pIRES2EGFP. The recombinant plasmid pIRES2EGFP-hTIM-3 was transfected into mammalian cells by lipofection , The expression of protein was analyzed by flow cytometry (FCM) and Western blot, and stable transfected cell lines were screened by FCM and selective medium. Results: The eukaryotic expression vector pIRES2EGFP-hTIM-3 was successfully constructed. After transfection with mammalian cells COS-7 and CHO, FCM and Western blot confirmed that TIM-3 was highly expressed on the cell membrane surface and stable transfected CHO cells Strain. Conclusion: The human TIM-3 eukaryotic expression vector was successfully constructed. TIM-3 was highly expressed on the surface of transfected mammalian cells and a stable transfected cell line was established. Which provided the conditions for the further study of the biological function of TIM-3 and its ligands and the preparation and screening of anti-human TIM-3 mAb.