人细胞色素c基因在大肠杆菌中的克隆和表达及活性测定

通过PCR方法 ,从人胎儿心肌基因组DNA中得到precytc基因 (有 1个内含子 ) ,剔除内含子后 ,得到细胞色素c基因的编码序列 .测序结果表明 ,该基因与GenBank中报道的人细胞色素c基因核苷酸顺序完全一致 .将其插入原核表达载体pET3a的NdeⅠ和HindⅢ位点之间构建pET3a cytc重组质粒 ,并成功转化入E .coliBL2 1(DE3)中 .经IPTG诱导表达后 ,15 %SDS PAGE分析 ,可观察到1条与细胞色素c蛋白分子量相符的电泳条带 .Western印迹结果显示 ,该条带与小鼠抗人cytc单克隆抗体IgG2b发生特异反应 ,证实为人细胞色素c的前体蛋白 .体外使血红素与该前体蛋白结合生成完整的人细胞色素c蛋白 ,其耗氧量在不同浓度具有与反应时间的线性关系 ,为研究人细胞色素c结构和功能关系奠定了基础

Cloning,Expression of Human Cytochrome c Gene in E.coli and Its Activity Assay

By the method of PCR a coding sequence of human cytochrome c gene has been obtained after deleting an intron included in precytochrome c gene which was extracted from human myocardium genome DNA. The result of sequencing showed that this gene had the same sequence as human cytochrome c gene reported in GenBank. The gene was inserted into expression vector pET 3a between Nde Ⅰand Hin dⅢ sites with correct open reading frame. pET 3a cyt c has been transformed into E.coli BL21(DE3). After induced by IPTG a special protein band appeared near the position of molecular weight 11.4 kD. Western blotting showed that the recombinant protein could react with a mouse monoclonal IgG 2b antibody raised against full length cytochrome c of human origin and confirmed that the recombinant protein was apocytochrome c. Heme was added to apocytochrome c successfully and holocytochrome c formation was monitored by optical spectra change of heme in 416 nm. A linear relation appeared between oxygen consumption and reaction time at different protein concentration of cytochrome c. This result will help us to discover the relation between the structure and the function of human cytochrome c.