幽门螺杆菌空泡形成细胞毒素A亚单位的克隆表达和P37抗体的制备  

Expression and Cloning of Helicobacter pylori Vacuolating Cytotoxin A Subunits and Preparation of Antibody against P37

作　　者：杨贵珍[1] 刘钟滨[2] 袁建平[3] 胡宝瑜[3] 时晓东[3] 郭晓奎[3] 

机构地区：[1]上海中医药大学病原生物学教研室,201203 [2]同济大学医学院病原生物学教研室 [3]上海交通大学医学院病原生物学教研室

出　　处：《胃肠病学》2006年第9期542-546,共5页Chinese Journal of Gastroenterology

摘　　要：背景:空泡形成细胞毒素A(VacA)是幽门螺杆菌(H.pylori)的重要致病因子,研究VacA的致病机制有助于进一步明确H.pylori的致病机制。目的:原核表达H.pyloriVacA亚单位P37和P58,制备P37多克隆抗体并分析其活性。方法:以聚合酶链反应(PCR)扩增H.pylorivacA基因的p37和p58片段,克隆入原核表达载体pQE31,构建重组质粒pQE31-p37和pQE31-p58,转化大肠杆菌(E.coli)M15,诱导蛋白表达。以镍-次氮基三乙酸(Ni-NTA)亲和层析纯化融合蛋白,以纯化P37蛋白免疫家兔,获取抗P37血清,以酶联免疫吸附测定(ELISA)检测抗体效价。以蛋白质印迹法(Westernblot)分析P37抗体与VacA的结合能力,观察P37抗体对VacA致胃癌细胞空泡化的抑制作用。结果:十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)显示,E.coliM15经诱导后表达相对分子质量分别为37kDa(1Da=0.9921u)和58kDa的P37和P58融合蛋白,经纯化后为单一蛋白。纯化P37蛋白免疫家兔后获得的抗P37血清抗体效价为1×106。P37抗体能中和VacA阳性H.pylori分泌的VacA,抑制VacA对胃癌细胞的空泡化作用。结论:本实验成功构建了原核表达载体pQE31-p37和pQE31-p58,获得了高纯度单一蛋白和高效价P37抗体,该抗体具有中和VacA毒性的作用。Background： Vacuolating cytotoxin A （VacA） is an important pathogenic factor of Helicobacter pylori （ H. pylori ）. Understanding the pathogenic mechanism of VacA is helpful for the elucidation of the pathogenesis of H. pylori. Aims： To express the subunits P37 and P58 of H. pylori VacA in prokaryotic cells and prepare polyclonal antibody against P37 and to assess the activity of the antibody produced. Methods： The fragments p37 and p58 of H. pylori vacA gene were amplified by polymerase chain reaction （PCR） and cloned into prokaryotic expression vector pQE31, resulting in two recombinant plasmids pQE31-p37 and pQE31-p58. Escherichia coil （E. coli） M15 was transfectcd with these recombinant plasmids respectively and was induced to express proteins. Fusion proteins produced were purified by Ni-nitrilotriacetic acid （Ni-NTA） affinity chromatography. Purified P37 protein was injected into rabbits and immune serum was collected. The antibody titer was measured by enzyme-linked immunosorbent assay （ELISA） and Western blot was used to analyze the binding ability of P37 antibody to VacA. The inhibitory effect on vacuolization of gastric cancer cells after neutralization of VacA with the P37 antibody was observed. Results： Fusion proteins P37 and P58 with relative molecular weight of 37 kDa （1 Da=0.9921 u） and 58 kDa respectively were expressed by transfected E. coli M15 and the proteins produced were demonstrated as homogenous proteins after purification by sodium dodecyl sulfate polyacrylamide gel clectrophoresis （SDS-PAGE）. The titer of antibody against P37 in the immune serum produced was 1 ×10^6. VacA produced by VacA-positive H. pylori could be neutralized by this antibody and the vacuolization effect of VacA on gastric cancer cells was inhibited. Conclusions： Two prokaryotic expression vectors pQE31-p37 and pQE31-p58 are constructed successfully and homogenous proteins are obtained after purification. High titer P37 antibody is obtained after inoculation of purified P37 pr

关 键 词：螺杆菌 幽门 空泡形成细胞毒素A 克隆 分子 抗体 

分 类 号：R392[医药卫生—免疫学]