耐碱锰超氧化物歧化酶基因(Mn-sodA)工程菌的构建及其表达条件  

Construction of Alkalotolerant Mn-superoxide Dismutase Engineered Strain and Its Expression Condition in Escherichia coli

作　　者：陈红漫[1] 裴雪亮[1] 孟鑫[1] 

机构地区：[1]沈阳农业大学生物科学技术学院,沈阳110161

出　　处：《农业生物技术学报》2009年第6期1103-1107,共5页Journal of Agricultural Biotechnology

基　　金：辽宁省农业综合开发办(No.02L315)资助

摘　　要：从Bacillus sp.110-2基因组上分离得到的耐碱锰超氧化物歧化酶基因(Mn-sodA),将其连接到原核表达载体pET-30a(+),得到重组载体pET-sodA,重组载体在大肠杆菌(Escherichia coli)BL21(DE3)中经异丙基硫代-β-D-半乳糖苷(IPTG)诱导得到表达。SDS-PAGE分析显示,融合表达产物的分子量大小为26.5kD。对含有sodA基因工程菌的表达情况研究表明,重组蛋白全部以可溶性形式存在;重组酶的比活力252U/mg,为原始菌株的2.1倍,目的基因在大肠杆菌中实现了高效表达;而且,重组酶还保留了野生型酶强的耐碱能力。BL21(pET-sodA)基因工程菌的构建提供了一种可利用的耐碱蛋白资源,同时探索了一种极端酶的生产方法。The alkalotolerant Mn-superoxide dismutase encoding gene （Mn-sodA） from Bacillus sp. 110-2 was obtained, and inserted in pET-30a （＋） to yield the recombinant expression vector pET-sodA. Over-expression of Mn-SOD in Escherichia coil BL21 （DE3） was achieved with pET-sodA, which was induced by Isopropyl-β-D-galactosidase （IPTG）. SDS-PAGE analysis showed that an over-expressed recombinant product about 26.5 kD, consistent with the molecular weight predicted from gene sequence was produced. The recombinant protein was soluble in BL21 （DE3）. The specific activity of the recombinant enzyme Mn-SOD was 252 U/mg, which was 2.1 times than that of wild type. It indicated that the expression of Mn-sodA was high in E.coli. Furthermore, the recombinant enzyme had a strong ability of alkali-resistance as wild enzyme. From the engineered strain-BL21 （pET-sodA）, an alkali-resistance protein resources and a method to product the enzyme with extreme ability can be obtained.

关 键 词：锰超氧化物歧化酶 耐碱性 极端菌 原核表达 

分 类 号：S188[农业科学—农业基础科学]