幽门螺杆菌感应蛋白CrdS的原核表达及生物信息学分析  

Prokaryotic expression and bioinformatics of responsive protein CrdS of Helicobacter pylori

作　　者：聂佳莹[1] 杨致邦[2] 唐磊[1] 黄进[1] 蒋英[1] 

机构地区：[1]重庆医科大学神经科学研究中心,重庆400016 [2]重庆医科大学基础医学实验教学中心病原生物学与免疫学实验室,重庆400016

出　　处：《中国生物制品学杂志》2013年第9期1228-1231,1236,共5页Chinese Journal of Biologicals

摘　　要：目的原核表达幽门螺杆菌(Helicobacter pylori,H.pylori)感应蛋白CrdS,并对其进行生物信息学分析,以探讨其酸适应的调控机制。方法从H.pylori 26695标准株全基因组DNA中PCR扩增编码CrdS蛋白的基因hp1364,插入原核表达载体pQE30,构建重组表达质粒pQE30-hp1364,转化大肠埃希菌(E.coli)XL1-blue,IPTG诱导表达重组CrdS蛋白。表达的重组蛋白经Western blot鉴定后,进行生物信息学分析。结果重组表达质粒pQE30-hp1364经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为46 000,表达量占菌体总蛋白的30%,主要以包涵体形式存在,可与Rabbit anti His-Tag Polyclonal Antibody特异性结合;生物信息学分析显示,CrdS的核苷酸序列和氨基酸序列与其他H.pylori菌株(HpG27、F30、B38)具有高度同源性,表面有许多亲水性区域,含多种易被磷酸化的氨基酸。结论成功原核表达了H.pylori CrdS蛋白,并进行了生物信息学分析,为进一步探讨CrdS的感应机制及通过阻断信号感应抗H.pylori感染的途径提供了实验材料。Objective To express the responsive protein CrdS of Helicobacter pylori in prokaryotic cells, analyze its bioinformatics and investigate its regulatory mechanism on acidic adaptation of H. pylori. Methods The hp1364 gene encoding CrdS protein was amplified by PCR from whole genomie DNA of H. pylori standard strain 26695, and inserted into prokaryotic expression vector pQE30. The constructed recombinant plasmid pQE30-hp 1364 was transformed to E. coli XLl-blue for expression under induction of IPTG. The expressed recombinant CrdS protein was identified by Western blot and analyzed for bioinformaties. Results Restriction analysis and sequencing proved that recombinant plasmid pQE30- hp1364 was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 46 000, contained about 30% of total somatic protein, mainly existed in a form of inclusion body, and showed specific binding to rabbit anti-His-tag polyclonal antibody. Bioinformatic analysis showed high homologies of nucleotide and amino acid sequences of CrdS to those of other H. pylori strains（HpG27, F30 and B38）. Many hydrophilic regions were presented on the surface of CrdS, which contained several kinds of amino acids easily to be phosphorylated. Conclusion The CrdS protein of H. pylori was expressed successfully in prokaryotie cells and analyzed for bioinformaties, which provided an experimental material for further study on responding mechanism of CrdS as well as the route in resist H. pylori infection through blocking signal response.

关 键 词：幽门螺杆菌 CrdS蛋白 原核细胞 基因表达 生物信息学 

分 类 号：R378.99[医药卫生—病原生物学] Q786[医药卫生—基础医学]