幽门螺杆菌黏附素HpaA重组蛋白的表达、纯化及晶体生长

发布时间：2019-01-26 09:00

【摘要】：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沘。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。
[Abstract]:Objective to construct the prokaryotic expression vector of Helicobacter pylori adhesin Hpa A and culture it with purified recombinant Hpa A protein for three-dimensional structural analysis, structural epitope analysis of Hpa A and study of adhesion antagonist. Methods the secondary structure of Hpa A protein sequence was analyzed by bioinformatics and the transmembrane region was predicted. The non-transmembrane region of hp0797 gene encoding Hpa A protein in Helicobacter pylori standard strain NCTC 26695 was amplified by PCR technique. The recombinant plasmid was constructed and p ET22b ()-r Hpa A, was induced by IPTG to express the recombinant protein in BL21 (DE3). The recombinant protein was purified by Ni ion affinity chromatography, anion exchange chromatography and gel filtration chromatography. The crystallization conditions were searched by Hampton Research crystallization kit and optimized systematically. The diffraction experiments were carried out at Shanghai Synchrotron radiation light 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 exists in the form of dimer in the solution, and the relative molecular weight of the SDS-PAGE analytical monomer is about 24000. The purity of the recombinant Hpa A protein is 95. The crystal size of the recombinant Hpa A protein is about 70 渭 m 脳 30 渭 m 脳 20 渭 m, and the diffractive resolution is 2.03 Bir. Conclusion Recombinant Hpa A single crystal was obtained by classical protein expression, purification and crystal culture techniques, which provides an important basis for three-dimensional structure analysis and structure-based study of Helicobacter pylori vaccine.
【作者单位】：第三军医大学全军免疫学研究所;第三军医大学药学系微生物与生化药学教研室;北京大学医学部人体解剖与组织胚胎学系;第三军医大学药学系药物化学教研室;
【基金】：863计划(2012AA02A407) 国家自然科学基金(81572810) 重庆市基础与前沿一般项目(CSTC2013JCYJA1037)
【分类号】：R392

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