重组人凋亡诱导因子基因的构建、表达及其对HeLa细胞的促凋亡作用

Gene Construction,Expression and The Apoptosis Inducing Effects on HeLa Cells of The Recombinant Apoptosis Inducing Factor

凋亡诱导因子 (apoptosis inducingfactor ,AIF)定位于细胞的线粒体膜间隙 .当凋亡信号刺激时 ,AIF分子从线粒体释放到胞浆 ,然后转位到细胞核内 ,引起染色体核周边凝集和DNA呈大片段断裂 (～ 5 0kb) .用RT PCR法分段克隆得到人全长AIF基因 ,经改造截去其N端线粒体定位信号编码序列 ,代之以不同长度的绿脓杆菌外毒素 (PE)转膜结构域序列 .把这些重组基因克隆入 pIRES2 EGFP真核表达载体 ,脂质体法转染HeLa细胞 ,通过荧光显微镜观察、共聚焦显微镜观察、电镜观察等方法检测了多种重组人AIF基因的表达及其对细胞生长的影响 .证明了重组人AIF基因的表达可引起HeLa细胞死亡 。

Apoptosis inducing factor (AIF) is a mitochondrial intermembrane space protein ubiquitously expressed in various kinds of cells. When death stimuli present, AIF is released from mitochondria to the cytoplasm and then translocated to the nucleus, inducing peripheral chromatin condensation and large scale fragmentation of DNA (～50 kb). The full length AIF gene was amplified by RT PCR firstly, then its N terminal mitochondrial localization sequence (MLS) was deleted, being replaced with PE transmembrane domain, and then the recombinant gene was inserted into the pIRES2 EGFP eukaryotic expression vector. After transfection into HeLa cells with lipofectamine, the expression of these recombinant AIF gene and their effects on HeLa cell growth were detected by fluorescent microscopy, confocal microscopy, and electron microscopy analyses. The result proved that the expression of the recombinant human AIF gene could induce HeLa cell death, which provided new strategy for killing cancer cells.