重组表达幽门螺杆菌致病岛cagl蛋白及其中和 - 第三军医大学学报.doc

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 钟情*1，2邹全明△2,郭刚2,章金勇2,张晓丽2 （1广州军区武汉总医院检验科,武汉 430070；2第三军医大学医学检验学临床微生物及免疫学教研室，重庆 400038） 摘要：目的 重组表达幽门螺杆菌（Helicobacter pylori，Hp），Hp黏附宿主细胞实验。方法 用PCR方法扩增出基因片段，将目的基因表达载体pET22b中诱导表达。采用亲和层析纯化蛋白，并以纯化蛋白为抗原免疫家兔，制备兔抗CagL多克隆抗体血清，多克隆抗体血清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6h时表达量最高8h时达到高峰，UVP扫描分析目的蛋白约占菌体总蛋白的35％～40％。SDS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。染色显微镜观察可见： Hp菌与HeLa细胞呈典型的聚集性黏附；中和后HpHeLa细胞未见黏附；DH5α的HeLa细胞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。 关键词：幽门螺杆菌；Ⅳ型分泌系统；CagL蛋白 IL-8；黏附阻断 中图法分类号： 文献标识码： The preliminary study on Recombinant CagL protein of Helicobacter pylori and its neutralization antibody Zhong Qing1，2，Zou Quan-ming2,Guo Gang2,Zhang Jin-yong2,Zhang Xiao-li2(1 Wuhan General Hospital,Guangzhou Military Area Command of Chinese People’s Liberation Army,Wuhan 430070、2 Department of clinical Microbiology and Immunology,Third Military Medical University,Chongqing 400038) Abstract：objective To study the function of cagL protein of recombinant Helicobacter pylori, preparation of rabbit anti-CagL specific polyclonal antibody and blocking the adhesion of Hp with host cells. Methods CagL gene was amplified by PCR, then constructed into prokaryotic expression vector pET-22b.Expressed protein was purified by affinity chromatography, and then immunized 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 Successfully constructed and purified recombinant CagL protein. 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 provides a good foundation for understanding Hp pathogenicity and colonization mechanism. Key words: Helicobacter pylori; CagL protein; Polyclonal antibody; IL-8