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| 酮古龙酸菌细胞色素c的表达、成熟及活性分析 | |
| 引用本文： | 程红,熊向华,赵岩,汪建华,张怡轩,张惟材.酮古龙酸菌细胞色素c的表达、成熟及活性分析[J].生物技术通讯,2011,22(4):488-492. |
| 作者姓名： | 程红 熊向华 赵岩 汪建华 张怡轩 张惟材 |
| 作者单位： | 1. 军事医学科学院,生物工程研究所,北京,100071;沈阳药科大学,辽宁,沈阳,110016 2. 军事医学科学院,生物工程研究所,北京,100071 3. 沈阳药科大学,辽宁,沈阳,110016 |
| 摘 要： | 目的:从酮古龙酸菌Y25中扩增细胞色素c基因,在大肠杆菌中表达、成熟并进行生物活性分析。方法:从酮古龙酸菌Y25基因组中PCR扩增细胞色素c基因,构建pET22b表达载体;从大肠杆菌BL21(DE3)中扩增细胞色素c成熟基因簇ccmABCDEFGH,连接到带有山梨糖脱氢酶组成型启动子的pBBR1MCS2-P200载体中;将构建的2个质粒共转化大肠杆菌BL21(DE3),经IPTG诱导表达后进行血红素染色检测;通过氧化还原光谱法对Ni柱亲和纯化的重组蛋白进行活性分析。结果:扩增得到1404 bp的细胞色素c基因及6481 bp的ccmABCDEFGH基因簇;重组菌株经IPTG诱导表达后行SDS-PAGE分析,可见相对分子质量为50×103的表达条带;血红素染色显示重组蛋白结合有血红素;经氧化还原光谱扫描,显示亲和层析纯化得到的目的蛋白有细胞色素c特征吸收峰。结论:从酮古龙酸菌Y25中扩增得到了细胞色素c基因,在大肠杆菌中进行了表达和成熟,表达蛋白具有细胞色素c生物活性。 |
| 关 键 词： | 细胞色素c 酮古龙酸菌 电子传递 血红素染色 |
Expression,Maturation and Activity Analysis of Cytochrome c of Ketogulonigenium vulgare |
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| CHENG Hong,XIONG Xiang-Hua,ZHAO Yan,WANG Jian-Hua,ZHANG Yi-Xuan,ZHANG Wei-Cai.Expression,Maturation and Activity Analysis of Cytochrome c of Ketogulonigenium vulgare[J].Letters in Biotechnology,2011,22(4):488-492. | |
| Authors: | CHENG Hong XIONG Xiang-Hua ZHAO Yan WANG Jian-Hua ZHANG Yi-Xuan ZHANG Wei-Cai |
| Institution: | CHENG Hong1,2,XIONG Xiang-Hua1,ZHAO Yan1,WANG Jian-Hua1,ZHANG Yi-Xuan2,ZHANG Wei-Cai1 1.Beijing Institute of Biotechnology,Beijing 100071,2.Shenyang Pharmaceutical University,Shenyang 110016,China |
| Abstract: | Objective: To amplify cytochrome c gene from Ketogulonigenium vulgare Y25,and express the maturation protein in E.coli for analyzing its bioactivity.Methods: The cytochrome c gene was amplified using PCR with K.vulgare Y25 genome as template,and was constructed into the expression vector pET22b.The ccmABCDEFGH gene cluster encoding cytochrome c maturation factors was amplified using PCR with E.coli BL21(DE3) genome as template,and was ligated into the vector pBBR1MCS2-P200 with constitutive promoter of sorb... |
| Keywords: | cytochrome c Ketogulonigenium vulgar electron transfer heme staining |
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