重组人铜锌超氧化物歧化酶在大肠杆菌中的表达研究  

Expression of Recombinant Human Superoxide Dismutase in Escherichia coli

作　　者：王岚[1,2] 刘新[1,2] 张帆[3] 肖纯凌 

机构地区：[1]辽宁省环境污染与微生态重点实验室,辽宁沈阳110034 [2]沈阳医学院基础医学院病原生物学教研室 [3]沈阳医学院基础医学院

出　　处：《沈阳医学院学报》2015年第2期70-73,共4页Journal of Shenyang Medical College

基　　金：沈阳医学院科技开发转化基金项目(No.20146069)

摘　　要：目的:构建人铜锌超氧化物歧化酶(h Cu,Zn-SOD)原核表达载体并诱导其表达,纯化及鉴定目的蛋白,为其临床应用奠定基础。方法:根据已报道的h Cu,Zn-SOD基因序列,采用RT-PCR技术从外周血单个核细胞(peripheral blood mononuclear cells,PBMC)中获得SOD c DNA序列。将所得的PCR产物插入原核表达载体p ET22b(+)中,重组质粒经酶切、PCR及测序鉴定正确后,将其转化至大肠杆菌Rosseta(DE3),通过异丙基硫代半乳糖苷(isopropyl thiogalactoside,IPTG)诱导表达出目的蛋白,经镍固定金属亲和层析纯化。采用黄嘌呤氧化酶法测定SOD生物学活性。结果:序列分析表明SOD基因成熟肽编码区含有465 bp与Gen Bank(X02317)中已报道序列一致的SOD核苷酸。经IPTG诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳分析显示表达的目的蛋白分子量约为19 k D,免疫印迹分析显示其与商品化的His-tag抗体呈特异性反应,但主要以包涵体形式表达,可溶性蛋白表达量很少。经Ni2+-NTA琼脂糖纯化获得SDS-PAGE电泳下单一条带。可溶蛋白酶活力为460 U/ml(SOD的酶比活力为630 U/mg)。结论:在大肠杆菌中获得了人源SOD的高效表达,为研究其生物学功能及广泛应用奠定了基础。Objective： To construct human copper zinc superoxide dismutase （hCu, Zn-SOD） prokaryotic expression vector and to purify and identify in order to lay the foundation of clinical application. Methods： RT-PCR technology was used to amplify SOD cDNA according to the reported sequence of hCu, Zn-SOD gene derived from peripheral blood mononuelear cells （PBMC）. The resulting PCR product was inserted into the prokaryotic expression vector pET22b （＋）. The recombinant plasmid was transformed into Escherichia coli Rosseta （DE3） after verifing by enzyme digestion, PCR and sequencing identification. The target protein was ex- pressed by IPTG induction and purified by nickel affinity immobilized metal chromatography. The biological activity of SOD was deter- mined by xanthine oxidase method. Results： Sequence analysis showed that 465 bp nucleotide in mature peptide encoding region was consistent with SOD sequence reported in GenBank （X02317）. After induced by IPTG and SDS-PAGE electrophoresis analysis, the recombinant protein was expressed and molecular weight was about 19 kD. But soluble protein expression was rarely and mainly in the form of inclusion body expression. The recombinant protein was purified by Ni2^-NTA agarose. Biological activity of soluble protease was 460 U/ml （SOD specific activity was 630 U/mg）. Conclusion： High expression of human SOD is obtained from Escherichia co- li, which laid the foundation for the study on its biological functions and wide application.

关 键 词：超氧化物歧化酶 大肠杆菌 表达 

分 类 号：Q786[生物学—分子生物学]