摘要
目的: 观察人截短型AIF(AIF△1 480 )基因的表达对HeLa细胞的促凋亡作用. 方法: 在人截短型AIF(AIF△1 120)基因克隆成功的基础上,进一步改造,构建截去AIF基因线粒体定位信号、FAD结合结构域和NADH结合结构域(1 480位氨基酸 )编码序列的人截短型AIF(AIF△1 480)基因.将其克隆入pIRES2 EGFP绿色荧光蛋白 (EGFP)共表达载体,用脂质体法转染HeLa细胞,通过荧光显微镜观察、电镜观察等方法,检测目的基因在转染细胞中的表达以及对转染细胞形态的影响.结果: 成功构建了人截短型AIF(AIF△1 480)基因的真核表达载体.转染细胞后,可检测到人截短型AIF(AIF△1 480)分子的表达.转染后 48h,可观察到表达人截短型AIF(AIF△1 480 )分子的细胞呈现典型的凋亡特征.结论: 人截短型AIF(AIF△1 480)基因的表达可诱导HeLa细胞凋亡.
AIM: To observe the expression of the truncated human apoptosis-inducing factor (AIF) gene and its apoptosis-inducing effect on HeLa cells.METHODS: After the truncated human apoptosis-inducing factor (AIF△1-120) gene was successfully cloned,the shorter truncated human AIF gene (AIF△1-480) was cloned,which was constructed by deleting the N-terminal mitochondrial location sequence (MLS) and the coding sequence of flavin adenine dinucleotide (FAD) binding domain and nicotinamide adenine dinucleotide (NADH) binding domain of AIF protein.The truncated human AIF gene(AIF△1-480) was inserted into the EGFP co-expression vector pIRES2-EGFP,and the pIRES2-EGFP-AIF△1-480 was then transfected into HeLa cells with LipofectAMINE.The expression of the truncated AIF gene and its effect on HeLa cell were detected by fluorescence microscope and electron microscope analysis.RESULTS: The eukaryotic expression vector pIRES2-EGFP containing the truncated human AIF(AIF△1-480)gene was successfully constructed.The AIF protein could be detected in the transfected HeLa cells.After transfection,typical apoptotic changes of the transfected HeLa cells and a mass of cell death were observed under electron microscope.CONCLUSION: The expression of the truncated human AIF(AIF△1-480)gene can induce apoptosis of the transfected human HeLa cells.
出处
《第四军医大学学报》
北大核心
2005年第6期484-487,共4页
Journal of the Fourth Military Medical University
基金
国家杰出青年科学基金资助项目(39925036 )
军队杰出中青年人才科研基金资助项目 ( 98J009 )
国家"863"计划资助项目(2004AA217071)
