截短型人Bid融合蛋白对HeLa细胞的促凋亡作用

Pro-apoptotic effect of a truncated human Bid fusion protein on HeLa cells

目的 :观察截短型人bid及其N端融合蛋白的表达对HeLa细胞的促凋亡作用 .方法 :通过反转录PCR方法获取人全长bid基因 ,再经PCR方法克隆得到截短型bid(trun catedbid ,tbid)基因及其N端融合蛋白基因插入pcDNA3真核表达载体 ,脂质体法转染HeLa细胞 ,通过间接免疫荧光 ,电子显微镜以及流式细胞仪等方法观察被转染细胞的生长及凋亡状况 .结果 :成功获得人bid基因 ,构建了tbid基因及tbid与绿脓杆菌外毒素 (PE)转膜结构域部分肽段的融合蛋白基因的真核表达载体 (pcDNA3 tbid6 1 ,pcDNA3 PE tbid6 1 ) .与对照组相比 ,在转染后 1 2～ 4 8h内两种基因的表达均导致HeLa细胞发生凋亡 ,且两者活性相当 .结论

AIM: To investigate the pro apoptotic effect of a truncated human Bid fusion protein on HeLa cells. METHODS: A full length bid gene was obtained by RT PCR. Then the full length bid gene was truncated by deleting its N terminal fragment to get a truncated bid (t bid ) gene, which was then fused with partial translocation domain sequence of pseudomonas exotoxin A (PE) to get a fusion protein gene. Both t bid gene and its fusion protein gene were respectively inserted into the pcDNA3 eukaryotic expression vectors , and transfected into HeLa cells. The apoptotic changes in the transfected cells were detected by indirect immunofluorescence technique, electron microscopy and flow cytometry analyses. RESULTS: The products, including full length bid gene, the genes and expression vectors of t bid and its fusion protein with part of the translocation domain of PE were successfully obtained. Compared with the control group, the expression of both genes displayed typical characteristics of apoptosis and had similar effects on HeLa cells. CONCLUSION: Both tBid and its fusion protein can effectively induce apoptosis of HeLa cells.