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目的 构建融合蛋白FADDdel GFP真核表达载体 pFADDdel GFP ,用该融合基因转染小鼠胰岛瘤细胞 (NIT) ,从而抑制死亡受体介导的细胞内凋亡信号传导 ,以探讨IDDM的发病机制及防治措施。方法 采用基因重组技术将FADDdel定向连接于真核表达载体 pEGFP N1 ,用脂质体将重组子pFADDdel GFP导入哺乳动物细胞NIT并检测其表达 ,采用FACS和MTT法检测特异性T细胞对转染重组子NIT引起的细胞毒效应。结果 转染重组子 pFADDdel GFP的NIT可见绿色荧光蛋白表达 ;pFADDdel GFP转染的NIT对特异性T细胞介导的细胞毒有明显抵抗。结论 成功构建 pFADDdel GFP融合基因并获稳定表达 ;pFADDdel GFP转染NIT可有效抑制死亡受体介导的细胞内凋亡信号传导。
Objective To construct a fusion protein FADDdel GFP eukaryotic expression vector pFADDdel GFP and use this fusion gene to transfect murine islet cells (NIT) to inhibit the death receptor-mediated signal transduction pathway in order to explore the pathogenesis of IDDM. Prevention. METHODS: FADDdel was ligated into eukaryotic expression vector pEGFP N1 using gene recombination technique. The recombinant pFADDdel GFP was introduced into mammalian cell NIT by lipofectamine and the expression was detected by MTT assay. Sub-NIT-induced cytotoxic effect. Results The green fluorescence protein (NIT) was expressed in NIT transfected with recombinant pFADDdel GFP. NIT transfected with pFADDdel GFP showed significant resistance to specific T cell-mediated cytotoxicity. Conclusion The pFADDdel GFP fusion gene was successfully constructed and stably expressed. Transfection of NIT with pFADDdel GFP could effectively inhibit the death receptor-mediated signal transduction of intracellular apoptosis.