人参铜锌超氧化物歧化酶原核表达载体的构建、表达及纯化  

Construction of prokaryotic expression vector,expression and purification of ginseng Cu /Zn superoxide dismutase

作　　者：林红梅[1] 王泽玉[2] 邵玥[2] 秦晓晔[2] 刘诗超[3] 张鑫[3] 杨利民[1] 

机构地区：[1]吉林农业大学中药材学院,吉林长春130118 [2]长春中医药大学附属医院,吉林长春130021 [3]长春中医药大学研发中心,吉林长春130117

出　　处：《中国中药杂志》2013年第23期4052-4055,共4页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81373937;31270371);国家科技支撑计划项目(2011BAI03B01)

摘　　要：研究提取人参叶中的总RNA,采用RT-PCR扩增人参叶Cu/Zn-SOD基因,构建pET-28(a)-Cu/Zn-SOD原核表达载体。使用Escherichia coli BL21(DE3)感受态细胞进行IPTG诱导表达。利用镍离子亲和层析进行纯化,并采用黄嘌呤氧化酶法测定目的蛋白的酶活。经RT-PCR扩增的人参Cu/Zn-SOD基因序列与GenBank数据库中公布的高丽参Cu/Zn-SOD基因序列同源性为99.00%。经IPTG诱导,目的蛋白的表达量约为44.42%,相对分子质量为16.30 kDa。纯化后的蛋白酶活可达到10 596.69 U·mg-1。通过分子生物学方法,成功构建了人参pET-28(a)-Cu/Zn-SOD原核表达载体,为进一步研究人参Cu/Zn-SOD生物学功能奠定了基础。The total RNA was extracted from ginseng leaves of Panax ginseng. The Cu/Zn-SOD gene was amplified via RT- PCR and the pET-28 （a）-Cu/Zn-SOD expression vector was constructed. The pET-28 （a）-Cu/Zn-SOD recombinant plasmid was trans- formed into Escherichia coli BL21 （DE3） competent cells and was induced by IPTG in order to select optimal induction of expression conditions. The target protein was purified by the nickel ions （ Ni ＋ ） affinity chromatography and the target protein enzyme activity was determinated by the xanthine oxidase method. The similarity of the Cu/Zn-SOD gene sequences and the Cu/Zn-SOD gene sequences of Korean ginseng in NCBI was 99.00%. The target protein expression level was about 44.42%, and the molecular weight was 16.30 kDa after the pET-28 （a）-Cu/Zn-SOD recombinants were induced by IPTG. The purified Cu/Zn-SOD protease activity reached 10 596.69 U · mg-7. The P. ginseng pET-28 （a） -Cu/Zn-SOD prokaryotic expression vector was built by the method of molecular biol- ogy, which provided the foundation for studying the Cu/Zn-SOD biology function. ：

关 键 词：人参 CU Zn—SOD 原核表达 

分 类 号：S567.51[农业科学—中草药栽培]