靶向线粒体绿色荧光蛋白表达载体的构建及初步应用

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...

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

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.