肺炎链球菌热休克蛋白Grp E的表达、纯化及热稳定性研究  

Expression,purification and thermal stability of heat shock protein Grp E from Streptococcus pneumonia

作　　者：张宏鹏[1] 赵沙沙[2] 龙小滨 吴爽[1] 白垒 罗淼 黄爱龙[3] 汪德强[3] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断重点实验室,重庆400016 [2]成都中医药大学附属医院检验科,成都610072 [3]感染性疾病分子生物学教育部重点实验室,重庆400016 [4]重庆市渝北区人民医院医学检验科,重庆401120

出　　处：《重庆医科大学学报》2014年第10期1448-1451,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:31240082);国家青年自然基金资助项目(编号:81301395);重庆市自然科学基金资助项目(编号:2011BB5124)

摘　　要：目的:肺炎链球菌可引起细菌性肺炎和脑膜炎、发热性菌血症等多种疾病。对肺炎链球菌热休克蛋白Grp E进行克隆表达,纯化及热稳定性测定,以便于进一步分析肺炎链球菌感染的分子机制。方法:利用PCR技术扩增Grp E基因,构建重组质粒p W28-Grp E,并在大肠杆菌(escherichia coli,E.coli)B834中表达,Ni2+-NTA亲和层析柱和DEAE阴离子交换层析柱纯化Grp E蛋白,Hiload Superdex 75凝胶层析柱分析在溶液中的聚集状态,热稳定性(thermal shift assay,TSA)法测定热稳定性。结果:1成功构建重组质粒p W28-Grp E,在表达菌E.coli B834中可溶性表达。2获得高纯度(95.0%以上)Grp E蛋白,发现该蛋白在溶液中以二聚体形式存在。3测定Grp E蛋白在p H 6.0、20 mmol/L盐浓度缓冲液中有较高热稳定性。结论:利用经典的蛋白质克隆表达纯化技术,获得了高表达量高纯度的Grp E蛋白,并初步测定其热稳定性为下一步蛋白晶体培养、三维结构解析及感染机制研究提供重要基础。Objective ：Streptococcus pneumonia is a major cause of several diseases. To express and purify the heat shock protein GrpE from Streptococcus pneumonia in order to uncover the infectious mechanism. Methods：Being amplified by PCR,the open reading frame of GrpE was subcloned into pW28 and expressed in escherichia coli（E.coli） B834. The bacterial protein was purified by nickel chro- matography column and anion-exchange chromatography column subsequently. The aggregation of GrpE in solution was analyzed by ge 1 filtration column（Hiload Superdex 75） and its thermal stability was characterized with thermal shift assay（TSA） method. Results： The GrpE protein was expressed in E.coli B834 as soluble. The purified protein with high purity of 95.0% exhibited homodimer in solutions. The recombinant GrpE protein presented relative stabilization in a solution at pH 6.0 and 20 mmol/L NaC1. Conclusion： The GrpE protein with high purity was expressed in E.coli B834 and its thermal stability was determined with TSA method.

关 键 词：肺炎链球菌 热休克蛋白Grp E 蛋白质表达纯化 热稳定性 

分 类 号：R378.14[医药卫生—病原生物学]