幽门螺杆菌NCTC11637株Hp1501蛋白的原核表达与纯化  

Prokaryotic Expression and Purification of Hp1501 Protein of Helicobacter pylori NCTC11637 Strain

作　　者：杨继文[1,2] 杨致邦[1] 敬保迁[2] 于拽拽[1] 熊玉霞[1] 王朝丽[2] 冯莉[2] 

机构地区：[1]重庆医科大学基础医学实验教学中心病原生物学与免疫学实验室神经科学研究中心,重庆400016 [2]川北医学院分子生物学研究所,四川南充637007

出　　处：《中国生物制品学杂志》2011年第12期1421-1424,共4页Chinese Journal of Biologicals

摘　　要：目的原核表达并纯化幽门螺杆菌(Helicobacter pylori,H.pylori)NCTC11637株Hp1501蛋白。方法 PCR扩增H.pylori NCTC11637株Hp1501基因编码功能区序列Hp1501a,定向克隆至pQE30载体,构建重组原核表达质粒pQE30-Hp1501a,转化E.coli XL1-blue,IPTG诱导表达,并分析重组蛋白的表达形式。用Ni-NTA His柱对重组蛋白进行纯化。结果重组表达质粒pQE30-Hp1501a经双酶切鉴定构建正确,测序分析表明插入基因序列完全正确;表达的重组蛋白相对分子质量约为37 000,表达量约占菌体总蛋白的21%,主要以包涵体形式存在,可与兔抗His单克隆抗体特异性结合;纯化的重组蛋白纯度达91%。结论成功原核表达并纯化了H.pylori NCTC11637株Hp1501蛋白,为H.pylori外膜蛋白的生物学功能和疫苗的研究奠定了基础。Objective To express the Hp1501 protein of Helicobacter pylori NCTC11637 strain in prokaryotic cells and purify the expressed product.Methods The Hp1501a sequence in encoding region of Hp1501 gene of H.pylori NCTC11637 strain was amplified by PCR and cloned into vector pQE30.The constructed recombinant plasmid pQE30-Hp1501a was transformed to E.coli XL1-blue and induced with IPTG,and the expressed recombinant protein was analyzed for form then purified by Ni-NTAHis column chromatography.Results Restriction analysis proved that recombinant plasmid pQE30-Hp1501a was constructed correctly.Sequencing result showed correct sequence of inserted gene.The expressed recombinant protein,with a relative molecular mass of about 37 000,contained about 21% of total somatic protein,mainly existed in a form of inclusion body,and showed specific binding to rabbit anti-His monoclonal antibody.After purification,the recombinant protein reached a purity of 91%.Conclusion The Hp1501 protein of H.pylori NCTC11637 strain was successfully expressed in prokaryotic cells and purified,which laid a foundation of study on biological function of H.pylori outer membrane protein and development of vaccine.

关 键 词：幽门螺杆菌 Hp1501基因 生物信息学 原核细胞 基因表达 纯化 

分 类 号：R378[医药卫生—病原生物学] Q786[医药卫生—基础医学]