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

摘要

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 pET 30a-Aj-SOD1 was constructed with pET30a(+ ) double enzyme digested by Nde Ⅰ /Xho Ⅰ and Aj-SOD1.pET30a-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 with 0.1 mmol/L IPTG after 8 h and detected by SDS-PAGE and pyrogallol autoxidation method.%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(A pos-tichopus 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:JX097096.1,459 bp),构建克隆载体pMD18-Aj-SOD1;在目的基因两端引入(XhoⅠ/N deⅠ)酶切位点,构建克隆载体pMD18-Aj-SOD1(XhoⅠ/N deⅠ).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0.1mmol/L IPTG诱导8h后,成功获得具有抗氧化活性的Aj-SOD1蛋白.