【摘要】 采用基因工程技术从发菜总DNA中克隆了一段的基因序列,该序列与基因库中已公布的编码地木耳(Nostoc commnue)超氧化物歧化酶的氨基酸序列同源性为97%。将该基因插入含T7启动子质粒pET-32中构建表达质粒pET-sod,然后将该表达质粒转入大肠杆菌BL21中进行蛋白表达,表达菌株用1 mmol.L-1IPTG诱导表达数小时后,产生较多的重组的蛋白,且该蛋白以可溶性蛋白形式存在。SDS-PAGE分析表明,在相对分子量约为22 kd的位置有一条明显蛋白质带。将诱导表达后的蛋白通过亲和层析的方法进行蛋白纯化;NBT光还原法测定表达产物的比活力,每毫克纯化蛋白约为2 550 U。对纯化后的蛋白进行高温胁迫研究,将该纯化蛋白在60℃高温下胁迫90 min后,其活性为原(未经胁迫)蛋白活性的85%。更多还原

【Abstract】 SOD gene was cloned from Nostoc flagelliforme and the amino acid sequence is 97% identical to that of Nostoc commune published.The gene was inserted into a constructed E.coli expression plasmid pETsod and the plasmid was transferred into expressing host BL21.Induced by 1 mmol·L-1 IPTG,the recombinant SOD of Nostoc flagelliforme was accumulated to a very high percent and the protein is exist as soluble protein.SDS-PAGE analysis revealed that the molecular weight of expression SOD of Nostoc flagelliforme was approximate 22 kd.Purified by Ni2+-resin column,the specific enzymatic activity was measured using NBT method and the calculated result of this purified enzyme is 2 550 U·mg-1. It was also found that after high temperature stress at 60℃ for 90 min,the specific enzymatic activity still retains 85%.更多还原