文档介绍：Caspase┐3融合蛋白基因的构建及其对HeLa细胞凋亡的诱导作用
作者:贾林涛,于翠娟,许彦鸣,朱峰,彭玮丹,苏成芝,王成济,杨安钢
【关键词】天冬氨酸蛋白酶类
关键词: 天冬氨酸蛋白酶类;脱噬作用;肿瘤;基因疗法
摘要:目的观察重构型caspase-3及其N-端融合蛋白的表达对HeLa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pcDNA3,脂质体法转染HeLa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HeLa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HeLa细胞发生凋亡.
Keywords:aspartic proteinases;apoptosis;neoplasms;gene therapy
Abstract:AIM To investigate the induction of HeLa cells to apoptosis by the expression of reversed caspase-3and its N-terminal fusion RT-PCR and bi-nant PCR were used to amplify human caspase-3gene and construct reversed caspase-3and its fusion protein cloning into expression vector pcDNA3and trans-fecting HeLa cells,we evaluated the growth condition and apoptosis of these cells by cell counting,electron microscope photographs,as well as MTT We suc-cessfully obtained human caspase-3gene,the genes and ex-pression vectors of reversed caspase-3and its fusion protein with part of the translocation domain of Pseudomonas exo-toxin A(PE).Compared with the control group,the expres-sion of both genes caused in a similar degree the decrease of living cell numbers and massive occurrence of