【摘要】 目的　构建表达幽门螺杆菌 (Helicobacterpylori,Hp)尿素酶B亚单位 (ureB)基因与大肠杆菌K - 12株hlyE基因融合表达的原核表达质粒 pTrc99A -hlyE 并表达。 方法　用PCR扩增hlyE基因 ,经酶切连接反应将其克隆入重组原核表达质粒并测序 ,与GenBank中的核酸和蛋白序列进行BLAST分析 ,重组的阳性克隆经IPTG诱导培养 ,SDS -PAGE电泳进行表达分析 ,薄层扫描分析融合蛋白的含量。结果　对重组质粒进行酶切和PCR检测 ,证明构建了携带hlyE及ureB基因的重组原核表达质粒 pTrc99A -ureB/hlyE ,核酸序列测定及同源性分析证实所构建的原核表达质粒pTrc99A -ureB/hlyE中所含的Hp -ureB及hlyE与GeneBank中的Hp -ureB和hlyE序列的同源性分别为 97 4 2 % (16 6 3/ 170 7)、99% (910 / 918)。重组细菌JM10 9可表达约 10 0kD的融合蛋白ureB/hlyE ,含量占全菌体蛋白含量的 15 5 %。 结论　构建并鉴定了原核表达质粒pTrc99A -hlyE并高效表达 ,为研究Hp口服疫苗奠定了基础更多还原

【Abstract】 Aim To construct recombinant prokaryotic expression plasmid pTrc99A ureB/hlyE and express in E coli Methods By genetic engineering methods,hlyE gene of E coli k 12 strain was cloned into a recombinant prokaryotic expression plasmid pTrc99A ureB the positive clones was screened by PCR?restriction enzyme digestion ,then sequenced analysis and aligned with the Hp ureB gene and hlyE gene provided by GeneBank The recombinant fusion protein ureB/hlyE was expressed in E coli and analyzed by SDS PAGE and thin layer screening Results The nucleotide sequences of ureB gene and hlyE gene in this recombinant prokaryotic expression plasmid had high homology with ureB gene of Helicobacter pylori (97 42%) and hlyE gene of E coli k 12 (99%) respectively The fusion protein ureB/hlyE was well expressed a 100 kD protein in JM109,which accoutered for 15 5% of total protein of recombinant bacterial Conclusion The recombinant prokaryotic expression plasmid pTrc99A ureB/hlyE has been constructed and identified;this work will help developing an oral recombinant vaccine against H pylori infection更多还原