幽门螺杆菌多表位重组原核表达工程菌的构建及表达特性研究

目的构建含有幽门螺杆菌尿素酶（UreI、UreB）及粘附素（HpaA）的多表位原核表达质粒pET28a(+)/ureI-ureB-hpaA〔pET28a(+)/IBA〕及相应的原核表达工程菌，研究其表达特性。方法通过生物信息学方法从ureI 、ureB和 hpaA基因中筛选T细胞和B细胞优势表位基因序列，人工合成并构建原核重组表达质粒pET28a(+)/IBA。经限制性内切酶（Nde Ⅰ，Xho Ⅰ）酶切鉴定及DNA测序鉴定后导入大肠杆菌BL21 (DE3) 。该工程菌经IPTG诱导后，用SDS-PAGE检测重组蛋白（rIBA）的表达情况，以抗幽门螺杆菌悉尼株（SS1株）特异卵黄抗体采用Western blot检测rIBA的抗原性。结果构建的多表位原核表达质粒pET28a(+)/IBA双酶切和测序鉴定与设计序列100%一致。工程菌经 IPTG 诱导后SDS-PAGE电泳显示在相对分子质量40×10 ³左右有一条明显蛋白条带，Western blot显示有相应位置特异反应条带。结论成功构建幽门螺杆菌多表位重组原核表达质粒pET28a(+)/IBA及相应原核表达工程菌，该工程菌表达的rIBA具有抗原性，表明该重组蛋白与幽门螺杆菌有高度相关性。

英文摘要:

Objective To construct the multi-epitope prokaryotic expression plasmid and appropriate engineering bacteria expressing the multi-epitope fusion protein of urea membrane channel protein (UreI), urease B subunit (UreB) and adhesin (HpaA) ofHelicobacter pylorithen study its microbiological characteristics. Methods The target sequence contains multi-epitope gene sequence of Helicobacter pylori were designed and synthesized, subsequently; it was subcloned into the expression vector pET28a (+), confirmed by restriction enzyme digestion and DNA sequencing. The fusion protein rIBA was expressed in E.coli Rosseta (DE3) and analyzed by Western blot. Results The plasmid of pET28a(+)/IBA was constructed successfully, confirmed by endonuclease digestion and sequence analyze. The expressed rIBA protein with relative molecular mass about 40×10 ³ and can be detected by Western blot. Conclusion The prokaryotic engineering bacteria expression multi-epitope of the Helicobacter pylori was constructed successfully. The recombinant protein rIBA expressed by the engineering bacteria can be identified by Sydney strain 1 of Helicobacter pylori (H. pylori SS1) specific antibody IgY, which demonstrated that the rIBA has high correlation with H.pylori SS1.

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