幽门螺杆菌尿素酶B亚单位的克隆表达及纯化-生物技术通报.PDFVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN ( ) 2015, 31 7 :220-225 幽门螺杆菌尿素酶 B 亚单位的克隆表达及纯化 闫锦锦  闫东明  邹雪  刘丹  彭超  苏亚南 （芜湖康卫生物科技有限公司，芜湖 241000） 摘 要 ： 为获得高纯度具有生物活性的尿素酶B 亚单位 （UreB）蛋白，利用PCR 方法扩增出ureB 目的基因，将其插入到 pET28a 载体中，构建表达质粒pET28a-ureB。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UreB 蛋白。采用 Q Sepharose High Performance 阴离子交换层析纯化，G-25 凝胶过滤层析脱盐，并通过SDS和免疫双扩散法对UreB 蛋白进行 鉴定。结果表明，该蛋白相对分子质量约为64 kD，与预期结果相符，脱盐后获得的UreB 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。 关键词 ： 幽门螺杆菌；尿素酶B 亚单位 ；UreB 蛋白 ；蛋白纯化 DOI ：10.13560/ki.biotech.bull.1985.2015.07.034 ， Cloning Expression and Purification of Urease B Subunit of Helicobacter pylori Yan Jinjin Yan Dongming Zou Xue Liu Dan Peng Chao Su Yanan （Wuhu Kangwei Biotechnology Co. ，Ltd. ，Wuhu 241000 ） Abstract: 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- was constructed successfully. The identified plasmid ureB 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 SDSand 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 th