葛仙米中SOD基因的克隆及在大肠杆菌中的表达

采用PCR技术从葛仙米（Nostoc sphaeroides）总DNA中克隆了一段基因序列，该序列与基因库中已公布的编码普通念珠藻（Nostoc commnue）超氧化物歧化酶的氨基酸序列同源性为98％,将该基因插入含T7启动子的质粒pET-32a（＋）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lmmol／L1PTG诱导表达数小时后，产生较多重组的蛋白，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22kd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2550U。

Cloning and expression of gene which encode SOD of Nostoc sphaeroides in Escherichia coli

A SOD gene was cloned from Nostoc sphaeroides and ito amino acid sequence was 98% identical to the published SOD amino acid sequence ofNostoc sphaeroides. We constructed a Escherichia coli expression plasmid pET-sod and transferred it into expressing host BI21. Induced by l mmol/L IPTG for several hours, the recombinant SOD of Nostoc sphaeroides was greatly produced and existed as soluble protein. SDS - PAGE analysis revealed that the molecular weight of expression SOD of Nostoc sphaeroides was approximate 22kd. Purified by Ni^2＋ - resin column and then detected by NBT method, specific activity of the enzymetic was 2550U per mg.