枯草芽孢杆菌Mn-SOD基因的克隆及原核表达  

Cloning of Mn-SOD gene from Bacillus subtilis and expression in Escherichia coli

作　　者：赵怡[1] 凌辉生[1] 李任强[1] 

机构地区：[1]暨南大学生物工程学系,广州510632

出　　处：《生态科学》2011年第2期174-177,共4页Ecological Science

基　　金：暨南大学自然科学基金(640073)

摘　　要：为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168 sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因。将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3)。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26 kD,占全菌蛋白的45.6%。改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^(-1),是对照组的4.84倍。枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础。In order to achieve the soluble expression ofMn-SOD gene in E. coli, Mn-SOD gene was amplified from the genomic DNA of Bacillus subtilis ATCC 9372 according to the sodA gene sequence ofB. subtilis 168. The cloned Mn-SOD gene was inserted into the expression vector pET-28a to be recombinant plasmid pET-28a-Mn-SOD and was then transformed for expression in E. coli BL21 （DE3）. The Mn-SOD protein with molecular weight of about 26 kD was expressed in E. coli after induction with isopropyl-fl-D - thiogalaetoside （IPTG）, which accounted for approximately 35.6 % of total bacterial protein. The enzyme activity of Mn-SOD was assayed with improved pyrogallic acid autoxidation method. The results showed that the specific activity of SOD in the total soluble protein was 51.09 U·mg^-1, and was 4.84 times of control group. Mn-SOD gene ofB. subtilis ATCC 9372 is first successfully expressed in E. coli BL21（DE3）, and its products possess high dissolubility and enzyme activity.

关 键 词：枯草芽孢杆菌 MN-SOD 原核表达 活力 

分 类 号：Q781[生物学—分子生物学]