幽门螺杆菌27 kDa外膜蛋白的基因克隆和特性鉴定  

Gene Cloning and Characterization of 27 kDa Outer Membrane Protein from Helicobacter pylo-ri PANG

作　　者：庞智[1] 朱红音[1] 徐蔚文[2] 萧树东[2] 

机构地区：[1]上海第二医科大学附属仁济医院 [2]上海市消化疾病研究所,200001

出　　处：《胃肠病学》2002年第5期265-269,共5页Chinese Journal of Gastroenterology

摘　　要：背景:幽门螺杆菌（H.Pylori）是全球人群中感染范围最广的致病菌,现己被公认为慢性胃炎和消化性溃疡的重要致病因子,并与胃腺癌和胃淋巴瘤的形成密切相关。而H.Pylori疫苗可能成为控制这一全球范围感染的有效措施,其研制和开发已成为目前研究的热点。目的:探索研制H.Pylori疫苗的新途径,对H.Pylori27kDa外膜蛋白（OMP27）进行基因克隆和特性鉴定。方法:培养和收集H.Pylori菌株NCTC11637,采用酚:氯仿抽提和纯化基因组DNA。分别设计引物P1和P2,并以该基因组DNA为模板,以聚合酶链反应（PCR）方法扩增OMP27基因片段。构建pQE30-OMP27重组表达载体时,pQE30质粒载体和纯化的PCR产物均用限制性内切酶Kpnl和HindⅢ双酶切,再用T4 DNA连接酶将双酶切后的目的基因片段OMP27重组于pQE30的相应酶切位点之间,连接产物转化大肠杆菌XL1-Blue。挑选转化克隆、提取质粒,并进行Kpnl和HindⅢ双酶切和PCR方法鉴定,1％琼脂糖凝胶电泳观察酶切和PCR扩增结果,经测序分析确认后,筛选出插入目的基因的阳性克隆,命名为pQE30-OMP27。挑取单个含重组质粒PQE30-OMP27的工程菌（XL1-Blue）阳性克隆,进行培养和IPTG诱导表达后,经SDS-聚丙烯酰胺凝胶电泳（PAGE）和Western blot进行蛋白表达和抗原性鉴定。Background: Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic, spiral bacterium which colonizes the mucosa of human stomach. The bacterium is the main cause of chronic active gas-tritis and of peptic ulcer, in particular duodenal ulcer, and plays a role in the development of gastric car-cinoma. A chronic H. pylori infection in humans usually induces a strong local and systemic immune re-sponse, which is not able to eradicate the bacteria. However, infection might be prevented by vaccina-tion and during recent years several membrane-associated antigens of H. pylori have been identified. Aims: To search the new protective antigens which can be used in a vaccine to prevent H. pylori infec-tion, a gene encoding the structural 27 kDa outer membrane protein (OMP27) from H. pylori strain NCTC11637 was cloned and characterized. Methods: Polymerase chain reaction (PCR) was used to am-plify the OMP27 gene from H. pylori chromosomal DNA. Recombinant plasmid (pQE30-OMP27) expressing OMP27 protein was constructed, and was transformed into E. coli strain XLl-Blue. The transformant col-ony was selected and identified, and the recombinant positive colony was induced with IPTG. Expression of OMP27 protein was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot. Results: Restriction enzymes cleavage analysis, electrophoresis analysis of PCR product of recombinant plasmid and sequencing showed that the target gene OMP27 had been successfully inserted into the pro-karyotic expression vector pQE30. The gene OMP27 was amplified to be 723 base pairs, which encoded objective polypeptide of 241 amino acid residues, corresponding to calculated molecular masses of 27 kDa. The SDS-PAGE analysis showed a dominant additional protein with molecular weight of 27 kDa, which accounted for 5% of total bacteria proteins. Western blot result showed that the recombinant protein could be recognized by specific-pathogen-free, female C57BL 6 mice sera infected with H. pylo-ri. Conclusions: The recombinant OMP27 p

关 键 词：PCR 基因表达 幽门螺杆菌27kDa 外膜蛋白 基因克隆 特性鉴定 

分 类 号：R392[医药卫生—免疫学]