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结核分枝杆菌ptpB的表达纯化及其相互作用蛋白的筛选

发表时间:2017-03-15  浏览量:1111  下载量:265
全部作者: 樊玲波,董春升,董元舒
作者单位: 苏州大学生物医学研究院
摘 要: 目的:构建、表达及纯化结核分枝杆菌(Mycobacterium tuberculosis,Mtb)ptpB(MptpB)蛋白,并筛选其在巨噬细胞中相互作用的蛋白。方法:研究构建pET28b-MptpB质粒,转化到大肠杆菌BL21,诱导表达后,使用Ni-His亲和层析法初步纯化,再用anti-His抗原抗体结合法进一步纯化出MptpB蛋白。利用His pull-down技术富集RAW264.7细胞中MptpB蛋白潜在的相互作用蛋白,通过SDS-聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)寻找差异性条带,并结合质谱进行蛋白鉴定。结果:成功构建并表达纯化出MptpB蛋白,通过质谱分析筛选出了其相互作用蛋白:Rvb1/Rvb2. 结论:初步探讨了在巨噬细胞中可能与MptpB相互作用的蛋白,为进一步研究MptpB在巨噬细胞中的作用奠定了实验基础。
关 键 词: 免疫病理学;MptpB;表达;纯化;His pull-down;相互作用蛋白
Title: Expression purification of Mycobacterium tuberculosis ptpB and screening for its interacting proteins
Author: FAN Lingbo, DONG Chunsheng, DONG Yuanshu
Organization: Institutes of Biology and Medical Sciences, Soochow University
Abstract: Objective: Construction, expression and purification of Mycobacterium tuberculosis (Mtb) ptpB (MptpB) and screening for its interacting proteins in macrophage. Methods: pET28b-MptpB plasmid was constructed and expressed in Escherichia coli BL21. The MptpB protein was purified by Ni-His affinity chromatography firstly and then anti-His antigen antibody method. The potential interacting proteins with MptpB were riched by His pull-down in RAW264.7 cells, and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The candidate proteins were identified by mass spectrometry. Results: The MptpB plasmid was constructed and the MptpB protein was purified successfully. In additon, potential interacting proteins (Rvb1/Rvb2) were screened by mass spectrometry analysis. Conclusion: We would provide clues to identifying the function of MptpB in macrophages by screening the interacting proteins with MptpB.
Key words: immunopathology; MptpB; expression; purification; His pull-down; interacting protein
发表期数: 2017年3月第5期
引用格式: 樊玲波,董春升,董元舒. 结核分枝杆菌ptpB的表达纯化及其相互作用蛋白的筛选[J]. 中国科技论文在线精品论文,2017,10(5):527-533.
 
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