摘要
目的 观察重构型 caspase- 3及其 N-端融合蛋白的表达对 He L a细胞的凋亡诱导作用 .方法 用反转录 PCR获取人 caspase- 3基因 ,用重组 PCR方法构建人重构型 caspase-3及其融合蛋白基因 ,将基因克隆入表达载体 pc DNA3,脂质体法转染 He L a细胞 ,通过细胞计数、电镜观察、MTT法等检测被转染细胞的生长及其凋亡状况 .结果 成功获得了 cas-pase- 3基因 ,构建了重构型 caspase- 3基因和重构型 caspase- 3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及其表达载体 ,与对照组相比 ,在转染后 1～ 4d内 ,两种基因的表达均导致 He L a细胞存活数减少 ,大量细胞发生凋亡 ,且二者活性相当 .结论 重构型 caspase- 3及其融合蛋白可以诱导转染的 He L
AIM To investigate the induction of HeLa cells to apoptosis by the expression of reversed caspase 3 and its N terminal fusion protein. METHODS RT PCR and recombi nant PCR were used to amplify human caspase 3 gene and construct reversed caspase 3 and its fusion protein genes. Following cloning into expression vector pcDNA3 and transfecting HeLa cells, we evaluated the growth condition and apoptosis of these cells by cell counting, electron microscope photographs, as well as MTT assays. RESULTS We successfully obtained human caspase 3 gene, the genes and expression vectors of reversed caspase 3 and its fusion protein with part of the translocation domain of Pseudomonas exotoxin A (PE). Compared with the control group, the expression of both genes caused in a similar degree the decrease of living cell numbers and massive occurrence of apoptosis. CONCLUSION Both reversed caspase 3 and its fusion protein can effectively induce HeLa cells to apoptosis.
出处
《第四军医大学学报》
北大核心
2001年第12期1057-1060,共4页
Journal of the Fourth Military Medical University
基金
国家杰出青年科学基金! (3 992 5 0 3 6)
军队杰出青年科学基金! (98J0 0 9)资助项目
