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目的:构建含线粒体编码的细胞色素氧化酶亚单位Ⅰ基因并使其转染人ρ~0SK-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Pmifo-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PCOⅠ-mito-RFP转染至ρ~0SK-Hep1细胞内,MTT法检测生长状况、Westernblotting检测细胞色素氧化酶亚单位Ⅰ((Cytochrome 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ρ~0SK-Hep1,MTT法测定发现重组载体COX Ⅰρ~0Sk-Hep1的生长速度快于其母本细胞株Sk-IHep1,低于线粒体DNA完全缺失的人肝癌细胞ρ~0Sk-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ρ~0Sk-Hep1,部分恢复了线粒体的功能。
OBJECTIVE: To construct the mitochondrial cytochrome oxidase subunit Ⅰ gene and to transfect human ρ ~ 0SK-Hep1 cells. The biological behavior and expression of the constructed vector were observed and detected. METHODS: Mitochondrial-encoded cytochrome C oxidase subunit Ⅰ was constructed by using the eukaryotic expression vector Pmifo-RFP. The expression vector PCOⅠ-mito-RFP was transfected into ρ ~ 0SK-Hep1 cells. The growth of the cells was detected by MTT assay. The expression of cytochrome oxidase subunit Ⅰ (CO Ⅰ) was detected by Western blotting.Results: The recombinant vector containing mitochondrial encoding cytochrome oxidase subunit Ⅰ was successfully constructed.Pmito-RFP-COX Ⅰ, The results showed that the growth rate of COX Ⅰρ ~ 0Sk-Hep1 was faster than its parental line Sk-IHep1, which was lower than that of human hepatoma cells ρ ~ 0Sk with complete deletion of mitochondrial DNA -Hep1 and Western blotting were used to detect the expression of COX Ⅰ protein.Conclusion: The recombinant plasmid Pmito-RFP-CO Ⅰ encoding mitochondrial cytochrome oxidase subunit Ⅰ was successfully constructed and found to grow faster than its parent The cell line SK-Hep1, which is lower than that of human hepatocarcinoma cell line ρ ~ 0Sk-Hep1 with complete absence of mitochondrial DNA, partially restored mitochondrial function.