幽门螺杆菌尿素酶B亚单位的克隆表达及纯化  

Cloning,Expression and Purification of Urease B Subunit of Helicobacter pylori

作　　者：闫锦锦 闫东明 邹雪 刘丹 彭超 苏亚南 

机构地区：[1]芜湖康卫生物科技有限公司,芜湖241000

出　　处：《生物技术通报》2015年第7期220-225,共6页Biotechnology Bulletin

基　　金：国家科技重大专项子课题项目(2014ZX09102042-002);安徽省科技攻关计划项目(1301041020)

摘　　要：为获得高纯度具有生物活性的尿素酶B亚单位(Ure B)蛋白,利用PCR方法扩增出ure B目的基因,将其插入到p ET28a载体中,构建表达质粒p ET28a-ure B。将鉴定正确的质粒转入大肠杆菌中培养,通过IPTG诱导表达获得Ure B蛋白。采用Q Sepharose High Per formance阴离子交换层析纯化,G-25凝胶过滤层析脱盐,并通过SDS-PAGE和免疫双扩散法对Ure B蛋白进行鉴定。结果表明,该蛋白相对分子质量约为64 k D,与预期结果相符,脱盐后获得的Ure B蛋白纯度为98.5%;免疫双扩散法证明该蛋白具有良好的生物活性和反应特异性。最终确定的纯化工艺,达到了一步纯化即得到高纯度、具有生物活性蛋白的目的,该纯化工艺简单、有效。In order to obtain high-purity and bioactive urease B subunit protein （ UreB ） , the fragments of ureB amplified by PCR was inserted into expression vector pET28a, and the recombinant plasmid pET28a-ureB was constructed successfully. The identified plasmid was transformed into Escherichia coli, which was induced to express by IPTG. The expressed protein UreB was purified by Q Sepharose High Performance anion exchange chromatography and desalted by G-25 gel filtration chromatography, then analyzed by SDS-PAGE and double immunodiffusion. The results indicated that the molecular weight of the protein was about 64 kD and its purity was 98. 5% after desalting, which was in accord with the prediction. Double immunodiffusion showed that protein UreB possessed a favorable bioactivity and specificity. The finalized purification process achieved the goal of 1-step purification may obtain high-purity and bioactive protein, and it was simpler than the existing purification process as well as effective.

关 键 词：幽门螺杆菌 尿素酶B亚单位 UreB蛋白 蛋白纯化 

分 类 号：R378[医药卫生—病原生物学]