摘要
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1mmol/LIPTG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.
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.
出处
《上海师范大学学报（自然科学版）》
2008年第3期301-304,共4页
Journal of Shanghai Normal University(Natural Sciences)
基金
上海市基础研究重点项目(03JC14057)
关键词
葛仙米
SOD基因克隆
诱导表达
Nostoc sphaeroides
gene cloning of SOD
induced expression
