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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：1880 下载量：537
| 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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