线粒体细胞色素氧化酶亚单位I表达载体的构建

摘要

Objective : Pmito-RFP-CO Ⅰ recombinant vector was constructed and transfected into p0SK-Hepl. Pmito-RFP-CO Ⅰ expression and biological behavior of transfected pOSK-Hep1 cells were assessed. Methods : Pmito-RFP-COX Ⅰ recombinant vector was constructed by molecular cloning and transfected into p0SK-Hep1 cells. The growth status of transfected p0SK-Hep1 cells was determined hy MTT, and expression of CO Ⅰ was assessed by Western blotting. Results: The p0SK-Hep1 cells transfected with pmito RFP-CO Ⅰ recombinant vector was found to grow faster than that of the parent cells but slower than that of p0SK-Hep1 c:ells( P ＜0. 01 ). CO Ⅰ expression was found in those transfected cells. Conclusion: The function of mitochondria may be recovered partially in the p0 SK-Hep1 cells transfected with pmito-RFP-CO Ⅰ .%目的:构建含线粒体编码的细胞色素氧化酶亚单位I基因并使其转染入PoSK-Hep1细胞,并对其构建的载体的生物学行为及表达进行观察及检测.方法:应用真核表达载体Pmito-RFP质粒构建含线粒体编码的细胞色素C氧化酶亚单位I,将表达载体PCO I-mito-RFP转染至PoSK-Hep1细胞内,MTT法检测生长状况、Western blotting检测细胞色素氧化酶亚单位I(Cytoehrome oxidase subunit I,CO I)蛋白的表达.结果:成功构建了含线粒体编码的细胞色素氧化酶亚单位I的重组载体Pmito-RFP-COX I,并以其转染PoSK-Hep1,MTT法测定发现重组载体COX I PoSk-Hep1的生长速度快于其母本细胞株Sk-Hep1,低于线粒体DNA完全缺失的人肝癌细胞PoSk-Hep1,Western blotting检测转染细胞可见COX I蛋白的表达.结论:成功构建了线粒体编码的细胞色素氧化酶亚单位I的重组载体Pmito-RFP-CO I,发现其生长速度快于其母本细胞株Sk-Hep1,低于线粒体DNA完全缺失的人肝癌细胞PoSk-Hep1,部分恢复了线粒体的功能.