文献类型：期刊文献

中文题名：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

英文题名：Cloning,expressing,Fermentation and purification of human copper/zinc superoxide dismutase cDNA

作者：施惠娟[1,2];范立强[1,2];陈长华[1,2];吴祥甫[1,2];袁勤生[1,2]

机构：[1]华东理工大学生物反应器工程国家重点实验室生物化学研究所;[2]中国科学院上海生物化学研究所

年份：1999

卷号：34

期号：5

起止页码：340

中文期刊名：中国药学杂志

外文期刊名：Chinese Pharmaceutical Journal

收录：CSTPCD;;EI(收录号：1999064706120);Scopus;北大核心:【北大核心1996】；CSCD:【CSCD2011_2012】；PubMed;

语种：中文

中文关键词：铜;锌;超氧化物歧化酶;CDNA;基因克隆

外文关键词：hCu,Zn SOD,RT PCR,gene cloning,gene expression,affinity chromatography

摘要：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。
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.