重组表达幽门螺杆菌致病岛CagL蛋白及其中和抗体初步研究

Preparation of recombinant CagL protein of Helicobacter pylori and its neutralization antibody

目的重组表达幽门螺杆菌(Helicobacter pylori,Hp)cagL基因,并制备兔抗多克隆抗体及阻断Hp黏附宿主细胞实验。方法用PCR方法扩增出cagL基因片段,将目的基因构建至表达载体pET22b中并诱导表达。采用亲和层析法纯化蛋白,以纯化蛋白为抗原免疫家兔,制备兔抗CagL多克隆抗体血清,并用多克隆抗体血清进行阻断Hp黏附宿主细胞实验及细胞因子IL-8活性分析。结果成功构建表达纯化了CagL重组蛋白,制备CagL蛋白兔抗多克隆血清,并初步验证了多克隆抗体血清在体外和Hp黏附作用的关系。与对照菌相比,重组菌CagL在25×103附近均出现新的蛋白表达条带,且诱导6 h时表达量最高。UVP扫描分析目的蛋白占菌体总蛋白的35%～40%。SDS-PAGE显示重组蛋白CagL主要以包涵体形式存在于超声沉淀中。ELISA结果显示兔抗CagL多克隆抗体的滴度为1∶16 000 EU。Giemsa染色后显微镜观察可见Hp菌与HeLa细胞呈典型的聚集性黏附,抗体中和后Hp与HeLa细胞未见黏附,加入DH5α的HeLa细胞也未见细菌黏附。结论多克隆抗体血清对Hp黏附定植有一定阻断作用,并且可以减少Hp促进细胞分泌IL-8的能力。

Objective To study the function of CagL protein of recombinant Helicobacter pylori （Hp） through preparing rabbit anti-CagL specific polyclonal antibody and blocking the adhesion of Hp with host ceils. Methods CagL gene was mnplified by PCR, then constructed into prokaryotic expression vector pET-22b. Ex- pressed protein of CagL was purified by affinity chromatography, and then used to immunize rabbits to prepare anti-CagL polyclonal antibody serum, so as to detect whether CagL polyclonal antibody serum can block the adhesion of Hp with host cells and analyze the activity of IL-8. Results Recombinant CagL protein was successfully constructed and purified. CagL polyclonal antibody serum played a stronger role in blocking the adhesion of Hp with host cells, and can induce host cells to secrete IL-8. Conclusion Our study suggests that CagL plays a role in Hp pathogenicity and colonization, and secretion of IL-8.