幽门螺杆菌全长hpaA基因克隆和在大肠杆菌中高效表达

Cloning and expressing in E. coli of Helicobacter pylori hpaA gene

为了构建幽门螺杆菌 (H .pylori)全长hpaA基因表达质粒 ,在大肠杆菌中表达重组HpaA(reHpaA)蛋白 ,我们用PCR扩增全长hpaA基因 ,经适当的酶切 连接反应将其连入原核表达质粒pTrc99A ,反应产物转化大肠杆菌JM10 5 ,用Amp(+)培养基筛选 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆 ,并用重组菌质粒进行hpaA基因测序。阳性克隆经IPTG诱导培养 ,用SDS PAGE电泳和Westernblot进行reHpaA表达分析和鉴定 ,用薄层扫描分析reHpaA含量。结果显示 ,重组质粒pTrc99A hpaA经PCR和酶切后均产生 783bphpaA基因。重组菌能表达约30kDreHpaA蛋白 ,含量占全菌体蛋白量的 5 1 99% ,Westernblot证实其有免疫反应性。重组H .pyloriHpaA表达质粒的成功构建及在大肠杆菌中高效表达的实现 ,为探索制备H .

To construct a recombinant Helicobacter pylori hpaA expression plasmid and express recombinant HpaA (reHpaA) in E. coli . A 783bp hpaA gene was ampified by PCR and linked to Nco I Sal I site of a procaryotic expression plasmid pTrc99A, the reaction product was then used to transform E. coli JM105 strain, the positive clones were screened by PCR and restriction enzyme digestion, and recombinant HpaA (reHpaA) was expressed in JM105 and analyzed by SDS PAGE and Western blotting. The results revealed that, confirmed by PCR and restriction enzyme digestion , a recombinant procaryotic expression plasmid pTrc99A hpaA was constructed, and reHpaA was well expressed in JM105 as a 30kD protein, which was 51.99% of total protein of recombinant JM105, and also its immunogenicity was confirmed by Western blot. These results suggested that a recombinant Helicobacter pylori hpaA gene expression plasmid was constructed and reHpaA was well expressed in JM105. This work will help to develop an recombinant oral vaccine against Helicobacter pylori .