海参铜锌超氧化物歧化酶原核表达载体的构建、表达及活性鉴定  

Construction,expression and activity identification of Apostichopus japonicus Cu/Zn-superoxide dismutase in E.coli BL21(DE3)

作　　者：车明月 穆贤 李学一 张齐[1] 丛丽娜[1] 牛庆昌 李成[1] 

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2018年第1期5-9,共5页Journal of Dalian Polytechnic University

基　　金：辽宁省教育厅一般科学研究项目(L2014217);辽宁省教育厅重点实验室项目(LZ2014029);辽宁省研究生教育创新计划项目(2014-154)

摘　　要：采用原核宿主大肠杆菌BL21(DE3)成功表达了具有活性的仿刺参铜锌超氧化物歧化酶(Apostichopus japonicus Cu/Zn-superoxide dismutase,Aj-SOD1)蛋白。利用Trizol法从海参肠组织中提取总RNA,通过反转录PCR扩增获得Aj-SOD1基因的编码序列(GenBank:JX097096.1,459bp),构建克隆载体pMD18-Aj-SOD1;在目的基因两端引入(XhoⅠ/NdeⅠ)酶切位点,构建克隆载体pMD18-AjSOD1(XhoⅠ/NdeⅠ)。将此载体与表达质粒pET30a(+)分别双酶切后连接,构建重组表达载体pET30a-Aj-SOD1。转化至大肠杆菌BL21(DE3),Kan+抗性筛选阳性转化子,获得基因工程菌BL21(DE3)-pET30a-Aj-SOD1。SDS-PAGE和邻苯三酚自氧化法检测发现,经0.1 mmol/L IPTG诱导8h后,成功获得具有抗氧化活性的Aj-SOD1蛋白。Apostichopus japonicus Cu/Zn-superoxide dismutase(Aj-SOD1) with activity was successfully expressed in prokaryotic host E.coli BL21(DE3).Total RNA was extracted from the intestinal tissue of Apostichopus japonicus by Trizol.Cu/Zn-SOD1 gene was amplified by RT-PCR to obtain the coding gene sequence of Aj-SOD1(GenBank:JX097096.1,459 bp)and pMD18-Aj-SOD1 was constructed.Cloning vector of pMD18-Aj-SOD1 with restriction sites of Nde Ⅰ/Xho Ⅰ was constructed. The recombinant expression vector of pET30 a-Aj-SOD1 was constructed with pET30 a(+)double enzyme digested by Nde Ⅰ/Xho Ⅰ and Aj-SOD1.pET30 a-Aj-SOD1 was transformed into E.coli BL21(DE3)competent cells and Kan+resistance screening positive transformants were selected.Aj-SOD1 proteins with antioxidant activity were successfully obtained by inducing with0.1 mmol/L IPTG after 8 hand detected by SDS-PAGE and pyrogallol autoxidation method.

关 键 词：仿刺参 铜锌超氧化物歧化酶 反转录PCR 抗氧化活性 

分 类 号：TS254[轻工技术与工程—水产品加工及贮藏工程] Q786[轻工技术与工程—食品科学与工程]