靶向线粒体绿色荧光蛋白表达载体的构建及初步应用  

Construction and Preliminary Application on the Vector Expressing Green Fluorescence Protein Target to Mitochondria

作　　者：杨健[1] 彭丽娟[1] 杨晓红[1] 

机构地区：[1]川北医学院微生物学与免疫学教研室,四川南充637007

出　　处：《川北医学院学报》2011年第5期397-400,共4页Journal of North Sichuan Medical College

基　　金：四川省科技厅项目(2009SZ0260);川北医学院重点实验室开放基金(KFJJ09-02)

摘　　要：目的:构建靶向线粒体绿色荧光蛋白(EGFP)表达载体,观测其在蛋白质细胞内定位中的应用价值。方法:化学合成细胞色素C氧化酶8A亚单位信号肽编码序列,克隆该序列到载体pEGFP-N1多克隆区Nhe I和Hind III位点得到质粒pMito-EGFP,经测序鉴定后,用线粒体探针mitotracker染色已转染该载体的Hela细胞,荧光显微镜检测EGFP的细胞定位。以EPEC/EspF和EPEC/EspFL16E感染经pMito-EGFP转染的Hela细胞,免疫细胞化学法和荧光显微镜法观测野生型EspF和突变型EspFL16E的分布。结果:测序表明融合表达载体构建成功,荧光显微镜及图像分析表明该载体表达的EGFP和mito-tracker共同定位于细胞线粒体;用其作为探针发现:野生型EspF定位于细胞线粒体,而突变型EspFL16E定位于胞浆中。结论:pMito-EGFP可作为线粒体的分子探针对靶向线粒体的蛋白进行亚细胞定位。Objective：To construct the plasmid pMito-EGFP which expressed enhancing green fluorescence protein（EGFP） target to mitochondria of mammalian cells and investigate the preliminary application.Methods： The plasmid pMito-EGFP was constructed by cloning the synthesized DNA sequence encoded signal peptide of cytochrome c oxidase subunit 8A between the Nhe I and Hind III site of pEGFP-N1 and determined by sequencing.Hela cells were stained with mitochondrial marker mitotracker after transfection with pMito-EGFP and the location of fluorescence was detected by fluorescence microscopy.Prior to infection with EPEC/EspF and EPEC/EspFL16E,Hela cells were transfected with pMito-EGFP,then the wide type EspF and mutant EspFL16E were stained by immunocytochemistry and the fluorescence was checked with fluorescence microscopy.Results： The vector pMito-EGFP was constructed successfully and EGFP was detected collocation in the mitochondria with mitochondrial marker mitotracker.Further investigation found wild type EspF injected by EPEC located in the mitochondria,nevertheless mutant EspF/L16E located in the cytoplasm.Conclusion： The vector pMito-EGFP can serve as a mitochondrial molecular probe for subcellular localization of protein target mitochondria.

关 键 词：线粒体 分子探针 致病性大肠杆菌 

分 类 号：R516.1[医药卫生—内科学]