结核分枝杆菌PPE60的GST融合蛋白在大肠杆菌中的表达和鉴定  

Expression and Identification of Mycobacterium Tuberculosis PPE60-GST Fusion Protein in Escherichia coli

作　　者：熊志红[1] 朱琰[1] 李仁德[1] 庄玉辉[1] 程小星[1] 

机构地区：[1]中国人民解放军第309医院结核病研究所,北京100091

出　　处：《实用预防医学》2012年第5期647-650,共4页Practical Preventive Medicine

基　　金：国家重大传染病专项基金资助项目(2008ZX10003-012)

摘　　要：目的构建结核分枝杆菌PPE60的GST融合蛋白表达载体,并在大肠杆菌中诱导表达融合蛋白。方法以结核分枝杆菌标准株H37Rv基因组DNA为模板,经PCR技术扩增目的基因,将目的基因克隆到pGEX-4T-1载体中,转化大肠杆菌DH5α,筛选阳性克隆。进行质粒的酶切鉴定和序列分析;对重组的大肠杆菌进行诱导表达,SDS-PAGE分析菌体蛋白,凝胶密度扫描分析目的蛋白的表达情况,Western-blot鉴定融合蛋白。结果正确构建了pGEX-PPE60融合表达载体,载体能在大肠杆菌中表达分子量约为70 kDa的重组蛋白,占菌体总蛋白的50%。该融合蛋白能与GST抗体产生阳性反应。结论成功构建了GST-PPE60融合蛋白的表达载体,并能在大肠杆菌中高效表达,为PPE60蛋白的功能研究奠定了基础。Objective To establish recombinant prokaryotic vector of Mycobacterium tuberculosis GST- PPE60 fusion gene. Methods PPE60 gene was amplified from genome of Mycobacterium tuberculosis H37Rv by PCR, then cloned into pGEX - 4T - 1 vector. The expression vector pGEX - PPE60 was transformed into Escherichia coli （ E. coli ） DH5α. Positive clones were screened. Plasmids were subjected to restriction enzyme digestion and nucleotide sequencing. Vector expression was induced in recombinant E. coli. Bacterial protein was analyzed by SDS- PAGE. Expression of target protein was determined by densitometry analysis. The GST - PPE60 fusion protein was identified by Western - blot. Results The restriction enzyme digestion analysis and nucleotide sequencing of recombinant vector demonstrated that the PPE60 gene had been exactly inserted into pGEX - 4T 1. SDS - PAGE analysis showed that the recombinant GST - PPE60 fusion protein was about 70 kDa in solu- tion form in E. coli DH5α. Thin layer scanning analysis showed the fusion protein amounted to 50% of total cell proteins. West- ernblot identified that it produced lxxsitive reaction with GST antibody. Conehtsions The recombinant prokaryotic vector pGEX - PPE60 is successfully established and GST - PPE60 fusion protein can be highly expreased in E. coli DH5α, which lays foundation for further function study of PPE60.

关 键 词：结核分枝杆菌 PPE60 GST-融合蛋白 表达与鉴定 

分 类 号：Q782[生物学—分子生物学]