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目的 构建含幽门螺杆菌 (Hp) ure A、ure B基因重组质粒 ,测定、分析其核酸序列及推定的氨基酸序列 ,在 E.coli中高效表达两基因 ,为检测试剂和疫苗研究提供基础依据和抗原。方法 PCR法扩增 Hp菌株 HPSH4的 ure A、ure B基因 ,分别与 p GEX- 2 T载体连接并转化大肠杆菌JM10 5 ,测定克隆基因的核酸序列并与 Gen Bank公布的国外 5株 Hp菌株 (2 6 6 95 ,HPK 5 ,J99,CPM6 30 ,M6 0 398)的 ure A、ure B基因作序列比较 ,以 IPTG诱导 ,ure A、ure B基因在 E.coli中高效表达。结果 ure A核酸同源性 96 .3%～ 98.1% ,氨基酸同源性 98.3%～ 10 0 % ;ure B核酸同源性95 .8%～ 98.0 % ,氨基酸同源性 96 .4 %～ 99.6 %。以 IPTG诱导 ,在 E.coli中表达 rure A融合蛋白5 5 6 0 0 D,rure B融合蛋白 85 5 0 0 D。结论 不同地区 Hp菌株的 ure A、ure B存在程度不同的变异 ,克隆并表达 HPSH4的 ure A、ure B与 Gen Bank已公布的多数 Hp菌株有极高同源性 ,在 E.coli以融合蛋白高效表达 rure A和 rure B
Objective To construct a recombinant plasmid containing ureA and ure B gene of Helicobacter pylori (H.pylori), to determine and analyze its nucleic acid sequence and putative amino acid sequence, to express two genes in E.coli efficiently and to provide a basis for the detection of agents and vaccines antigen. Methods The ure A and ure B genes of Hp strain HPSH4 were amplified by PCR and ligated into pGEX-2 T vector and transformed into E. coli JM10 5 respectively. The nucleotide sequence of the cloned gene was determined and compared with 5 strains of Hp strain The ure A and ure B genes of HPK 5, J99, CPM6 30 and M6 0 398 were sequenced and induced by IPTG. The ure A and ure B genes were highly expressed in E. coli. Results ure A nucleic acid homology 96.3% ~ 98.1%, amino acid homology 98.3% ~ 100%; ure B nucleic acid homology 95.8% ~ 98.0%, amino acid homology 96.4% ~ 99.6 %. Induced by IPTG, expressed in E. coli rure A fusion protein 56000D, rure B fusion protein 8550D. Conclusion ure A and ure B of Hp isolates in different regions have different degrees of variation. Ure A and ure B, which are cloned and expressed HPSH4, have very high homology with the majority of published H. pylori strains in Gen Bank. They are highly expressed in E. coli with the fusion protein rure A and rure B