发菜中超氧化物歧化酶基因的克隆及在大肠杆菌中的表达

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^-1 IPTG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%。

Cloning and Expression of Gene which Encode SOD of Nostoc flagelliforme in E. coli

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 pET-sod 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%.