Yu Cui-juan, Meng Yan-jing, Zhao Jing, Wang Zhi, Wang Cheng-ji, Yang An-gang
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2003 Jan;19(1):10-3.
To observe the expression of the truncated human apoptosis-inducing factor (AIF) gene and its apoptosis-inducing effect on HeLa cells.
Full-length human AIF gene was cloned by RT-PCR, then the truncated AIF gene was constructed by deleting the N-terminal mitochondrial location sequence (MLS), and inserted into the EGFP co-expression vector pIRES2-EGFP. After being transfected into HeLa cells with Lipofectamin, the expression of the truncated AIF gene and its effect on HeLa cells morphology and growth condition were detected by fluorescence microscope, immunohistochemical staining, indirect fluoroimmunoassay and electron microscope analysis.
The eukaryotic expression vector pIRES2-EGFP containing of truncated human AIF gene was constructed successfully. The AIF protein could be detected in the transfected HeLa cells. After transfection, typical apoptotic feature of the transfected HeLa cells and a mass of cell death were observed under electron microscope.
The expression of the truncated human AIF gene can induce apoptosis of the transfected human HeLa cells.
观察截短型人凋亡诱导因子(AIF)基因的表达及其对HeLa细胞的凋亡诱导作用。
通过RT-PCR克隆人AIF基因全长,然后删除N端线粒体定位序列(MLS)构建截短型AIF基因,并将其插入EGFP共表达载体pIRES2-EGFP。用Lipofectamin转染HeLa细胞后,通过荧光显微镜、免疫组织化学染色、间接荧光免疫测定和电子显微镜分析检测截短型AIF基因的表达及其对HeLa细胞形态和生长状况的影响。
成功构建了含截短型人AIF基因的真核表达载体pIRES2-EGFP。在转染的HeLa细胞中可检测到AIF蛋白。转染后,在电子显微镜下观察到转染的HeLa细胞具有典型的凋亡特征且有大量细胞死亡。
截短型人AIF基因的表达可诱导转染的人HeLa细胞凋亡。