人铜锌超氧化物歧化酶基因的克隆表达及活性鉴定  

Cloning Expression and Activity Detection of Human Copper Zinc-superoxide Dismutase Gene

作　　者：李雪莲[1] 武峰[2] 史兆兴[3] 

机构地区：[1]河南中医学院基础医学院,郑州市450008 [2]郑州大学基础医学院,郑州市450052 [3]中国人民解放军军事医学科学院生物工程研究所,北京市100071

出　　处：《医药论坛杂志》2006年第16期7-9,11,共4页Journal of Medical Forum

摘　　要：目的获得高表达人铜锌超氧化物歧化酶(hCu/Zn-SOD)的工程菌。方法采用RT-PCR技术从人胃组织总RNA中分离扩增了hCu/Zn-SOD的cDNA序列,将该基因重组到T7启动子控制下的分泌型表达载体pET22b(+)中,构建了表达质粒pETSOD,并转化大肠杆菌BL21(DE3)进行诱导表达。采用SDS-PAGE、蛋白质免疫印迹分析其活性。结果该工程菌可高表达一相对分子质量大约16kD的蛋白,与抗人Cu/Zn-SOD多克隆抗体有特异的免疫反应,表达量可达菌体可溶性蛋白的20%以上,且具有特异性SOD酶活性。结论该菌株分泌表达了有天然酶活性的hCu/Zn-SOD,为大量制备hCu/Zn-SOD和下一步研究工作奠定了基础。Objective To obtain the engineering E. coli for high expression of human copper,zinc- superoxide dismutase gene. Methods The cDNA encoding humanh Cu/Zn -SOD was amplified by RT - PCR using the total mRNA of human stomach as the template. The cloned hCu/Zn - SOD cDNA was ligated into expression vector pET22b（ ＋ ） under T7 promotor, then it was transformed into E. coli BI21 （ DE3 ） and induced. Then we analysised the total protein by 15% SDS - PAGE and Western - blot. SOD activity was assayed by the NBT photoreduction. Results The recombinant E. coli expressed the predicted 16kD human Cu/Zn - SOD, and its amount was over 20% of total proteins of the the bacteria in soluble form, which had specific SOD immunoreac.tivity and activity. Conclusion The engineering E. coli expressing hCu/Zn - SOD was established. The study lay a good basis for large scale purification and further practical research of hCu/Zn -SOD.

关 键 词：铜锌超氧化物歧化酶 分泌表达 大肠杆菌 

分 类 号：Q55-34[生物学—生物化学]