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论文摘要

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

The Research on the Construction and Characteristics of Recombinant Engineering Bacteria with Multi-epitope ofHelicobacter pylori

作者:丁娜娜, 杨靖, 潘兴等

Author:DING Na-na, YANG Jing, PAN Xing.et al

收稿日期:          年卷(期)页码:2014,45(3):367-370

期刊名称:四川大学学报(医学版)

Journal Name:JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION)

关键字:幽门螺杆菌 表位 尿素酶 粘附素 工程菌

Key words:Helicobacter pylori Epitope Urease Adhesin Engineering bacteria

基金项目:

中文摘要

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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