人EC-SOD的克隆及高效表达

Cloning and High Expression of Human EC-SOD cDNA

将编码人胞外超氧化物歧化酶 ( EC- SOD)成熟肽的 c DNA插入含 T7启动子的质粒p ET- 2 8a( + )中构建表达质粒 p ET- EC- SOD,表达菌株用 1 mmol/ L异丙基硫代 - β- D-半乳糖苷( IPTG)诱导表达 3h～ 5 h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 2 6%以上。经纯化和复性后的EC- SOD比活为每毫克纯化酶蛋白 1 2 0 0 U。将 EC- SOD c DNA基因插入昆虫杆状病毒供体质粒p Fast Bac HTb中 ,重组杆状病毒 Bac HT- EC- SOD转染粉纹夜蛾 Tn- 5 Bl- 4细胞 ,经扩增后在细胞内进行表达。SDS- 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 2 8ku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 2 60

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.