人EC-SOD的克隆及高效表达

将编码人胞外超氧化物歧化酶（EC-SOD）成熟肽的cDNA插入含T7启动子的质粒pET-28a( )中构建表达质粒pET-EC-SOD，表达菌株用1mmol/L异丙基硫代-β-D半乳糖苷（IPTG）诱导表达3h-5h后，产生较多的重组人EC-SOD，并形成包含体。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，表达的重组蛋白占菌体可溶性蛋白质的26%以上。经纯化和复性后的EC-SOD比活为每毫克纯化酶蛋白1200U。将EC-SOD转染粉纹夜蛾Tn-5Bl-4细胞，经扩增后在细胞内进行表达。SOS-PAGE分析结果表明，粉纹夜蛾细胞中表达一相对分子质量约为28ku的特异蛋白质带，Western blot分析表明，该特异条带即为EC-SOD蛋白，连苯三酚自氧化法测得表达产物比活为每毫克细胞裂解物260U。

Cloning and High Expression of Human EC-SOD cDNA

The gene encoding human EC SOD mature peptides ( EC SOD cDNA) was inserted into E.coli expression plasmid pET 28a(+) which contained the T7 promotor, then it was transformed into E.coli BL21(DE3). After induced with 1mmol/L IPTG, the recombinant human EC SOD was highly expressed as inclusion body. SDS PAGE analysis revealed that recombinant EC SOD was cumulated up to 26% of total soluble protein of E.coli cells. After purifying with Ni 2+ IDA Sepharose 6B column, denaturing and refolding, the specific enzymatic activity of recombinant human EC SOD was 1 200U every 1mg purified products. Also, the EC SOD cDNA was inserted into the donor plasmid pFastBacHTb. After transposition, transfection and amplification, the recombinant bacularviruses were infected Tn cells where EC SOD was highly expressed. SDS PAGE and Western blot analysis revealed that the molecular weight of expression human EC SOD was 28 ku, its specific activity was 260U every 1mg Tn cell lysates by pyrogallol autoxidation assay.