3α-羟类固醇脱氢酶基因的质粒载体构建和表达  

Cloning and expression of 3α-hydroxysteroid dehydrogenase gene in E.coli

作　　者：柯叶芳[1] 卢根杰[1] 侯玲玲[1] 吴锐浩[1] 李东[2] 张洪勤[2] 李佩珍[2] 应俊[2] 

机构地区：[1]温州医学院检验医学院,浙江温州325035 [2]温州医学院生物实验教学中心,浙江温州325035

出　　处：《中国微生态学杂志》2009年第9期822-824,832,共4页Chinese Journal of Microecology

摘　　要：目的实现3α-羟类固醇脱氢酶基因在大肠埃希菌中的高可溶性表达。方法从土壤中分离睾丸酮丛毛单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶(3α-HSD)基因,将它克隆到原核表达载体上进行诱导表达。提取细菌总蛋白进行SDS-PAGE分析并测定酶活性。结果经核苷酸序列测定和酶切鉴定结果表明,成功地构建了重组质粒,IPTG诱导表达后,获得融合蛋白,SDS-PAGE初步测定目的蛋白的相对分子量约为29kDa,与预期理论值一致;酶活性测定结果表明菌体可溶性总蛋白HSD酶比活性为142.81 U/mg,是对照BL21的12.97倍。结论该研究成功地构建了3α-羟类固醇脱氢酶基因高效原核表达系统,为利用基因工程手段大量制备3α-HSD的工作奠定了基础。Objective To achieve the overexpression of 3α--hydmxysteroid dehydrogenase（3α-HSD） gene in E. coli and better enzyme activity of the expressed product. Method 3α--hydroxysteroid dehydrogenase gene was amplified by PCR from a wild Comamonas testosteroni. The PCR product was cloned into pUC19-T vector and sequenced. The 3α-HSD prokaryotic expression system was constructed with plasmid pET28α as the vector and E. coli BI21 as the host bacteria. The fusion protein with an N-terminal His-tag sequence was purified in one step using metal chelate affinity chromatography to homogeneity on a Ni2 ＋-Sepharose column. Result The SDS-PAGE analysis revealed that the molecular weight of the re- combinant protein was about 29 kDa. The enzyme activity detection showed that the specific activity of the expressed prod- uct in the total soluble protein was 1428.49 U/rag, 129.74 times of that of E. coli BL21. Conclusion The soluble expres- sion of 3α-HSD gene in E. coli was successful.

关 键 词：大肠埃希菌 3α-HSD 原核表达 

分 类 号：R446[医药卫生—诊断学]