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目的构建幽门螺杆菌黏附素Hpa A的原核表达载体,并用纯化的重组Hpa A蛋白进行晶体培养,为其三维结构解析、基于结构的Hpa A抗原表位分析和黏附拮抗剂研究奠定基础。方法用生物信息学方法分析Hpa A蛋白序列二级结构并预测跨膜区,利用PCR技术扩增幽门螺杆菌标准株NCTC 26695编码Hpa A蛋白的hp0797基因非跨膜区片段,构建重组质粒并p ET22b(+)-r Hpa A,在大肠杆菌BL21(DE3)中IPTG诱导表达重组蛋白,经Ni离子亲和层析、阴离子交换层析、凝胶过滤层析纯化蛋白并分析其聚集状态。用Hampton Research结晶试剂盒搜索结晶条件,并系统优化,在上海同步辐射光源进行衍射实验。结果成功构建了重组质粒p ET22b(+)-r Hpa A,并在大肠杆菌BL21(DE3)中可溶性表达;重组Hpa A蛋白在溶液中以二聚体形式存在,SDS-PAGE分析单体相对分子质量约24 000,HPLC分析纯度达95%;培养出晶形较好的重组Hpa A蛋白单晶,大小约70μm×30μm×20μm,衍射分辨率2.03魡。结论利用经典的蛋白质表达、纯化技术和晶体培养技术,获得了衍射能力较强的重组Hpa A蛋白单晶,为其三维结构解析和基于结构的幽门螺杆菌疫苗研究提供了重要基础。
Objective To construct a prokaryotic expression vector for Helicobacter pylori adhesin Hpa A, and to use the purified recombinant Hpa A protein for crystal culture, which laid the foundation for its three-dimensional structure analysis, structural Hpa A epitope analysis and adhesion antagonist study. Methods The secondary structure of Hpa A protein sequence was analyzed by bioinformatics method and the transmembrane region was predicted. PCR was used to amplify the non-transmembrane region of hp0797 gene encoding Hpa A protein by NCTC 26695 and construct recombinant plasmid p ET22b (+) - r Hpa A, recombinant protein was induced by IPTG in E.coli BL21 (DE3), purified by Ni ion affinity chromatography, anion exchange chromatography and gel filtration chromatography. The crystallization conditions were searched by Hampton Research Crystallization Kit, and the system was optimized. The diffraction experiment was carried out in Shanghai synchrotron radiation source. Results The recombinant plasmid p ET22b (+) - r Hpa A was successfully constructed and expressed in E.coli BL21 (DE3). The recombinant Hpa A protein existed as a dimer in solution. SDS-PAGE analysis of the relative molecular weight The mass was about 24 000 and the HPLC purity was 95%. The recombinant Hpa A single crystal with good crystal shape was obtained. The size was about 70μm × 30μm × 20μm and the diffraction resolution was 2.03%. Conclusions The recombinant Hpa A single crystal with strong diffraction ability was obtained by classical protein expression, purification and crystal culture techniques, which provided an important basis for the study of three-dimensional structure and structure-based H. pylori vaccine.