人铜锌超氧化物歧化酶cDNA的克隆、表达、发酵及纯化工艺研究

 目的:用基因工程的方法获得人铜锌超氧化物歧化酶(hCu,Zn-SOD)的高产菌,通过发酵培养,最后纯化获得大量廉价的高纯度的hCu,Zn-SOD?方法:用逆转录-聚合酶链反应(RT-PCR),扩增了人铜锌超氧化物歧化酶(hCu,Zn-SOD)的cDNA,将此正确测定的cDNA重组到分泌型表达载体pET-22b(+)中,重组质粒在大肠杆菌BL21(DE3)中用5L全自动发酵罐表达hCu,Zn-SOD,表达产物用亲和层析一步法进行纯化?结果:hCu,Zn-SOD表达产物占菌体总蛋白的30%,发酵水平酶活力可达950u·ml^-1培基,比活为1400u·mg^-1,经亲和层析后纯度可达88%,活力回收率大于80%,比活大于5000u·mg^-1?结论:我们开展人重组SOD的研究不仅大大丰富了应用酶学的内容,而且积极开展应用研究,实现产业化,意义重大?

Cloning,expressing,Fermentation and purification of human copper/zinc superoxide dismutase cDNA

OBJECTIVE:To obtain high purified and cheap human copper/zine-superoxide dismutase(hCu,Zn-SOD) with genetic engineering and affinity chromatography.METHODS:The cDNA encoding hCu,Zn-SOD was amplified from the human liver total RNA by RT-PCR and sequenced.hCu,Zn-SOD cDNA was then ligated into expression vector pET-22b(+) under T7 promotor.The recombinant hCu,Zn-SOD was highly expressed in E.coli BL21(DE3)with a 5L totally automatic fermentation tank.The products were purified with affinity chromatography.RESULTS:The recombinant hCu,Zn-SOD was up to 30% of the total protein of the bacteria in soluble form.The enzyme activitives was 1400u·mg^-1 prot.After purified with affinity chromatography,the purity of hCu,Zn-SOD was 88%,the rate of recovery was up to 80%,and the specific activity was more than 5000u·mg^-1 protein.CONCLUSION:The study of human recombinant Cu,Zn-SOD not only enrichs the content of applied enzynology,but also actively develops its application.