| 分枝杆菌中表达重组蛋白的新载体pMSL的构建 |
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| 引用本文： | 叶子坚,刘冲,陈效友,昌增益.分枝杆菌中表达重组蛋白的新载体pMSL的构建[J].江西农业大学学报,2005,27(5):753-758. |
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| 作者姓名： | 叶子坚 刘冲 陈效友 昌增益 |
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| 作者单位： | 萍乡高等专科学校,生化系,江西,萍乡,337000;清华大学,生命科学与技术系,北京,100084;北京市结核病与胸部肿瘤研究免疫所研究室,北京,101149;北京大学,生命科学院,北京,100871 |
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| 基金项目： | 国家重点基础研究发展规划项目（No.G1999075607）和国家自然科学基金（No.30270289） |
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| 摘 要： | 为了能够利用耻垢分枝杆菌表达和纯化结核分枝杆菌蛋白，构建了大肠杆菌一分枝杆菌穿梭质粒pMSL。该质粒包括来源于牛型分枝杆菌hsp60基因启动子序列，目的蛋白插入的克隆位点和用于目的蛋白表达和定位进行跟踪的绿色荧光蛋白质（EGFP）基因，在目的蛋白插入位点和EGFP基因之间整合了凝血酶识别位点，便于融合蛋白纯化后目的蛋白与EGFP分离，在EGFP基因后面融合有6个组氨酸的密码子。结果表明，pMSL质粒能够在耻垢分枝杆菌中有效地表达绿色荧光蛋白，通过6个组氨酸尾，利用Ni—NTA亲和层析柱很好地被纯化。
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| 关 键 词： | 结核分枝杆菌 pMSL质粒 绿色荧光蛋白 表达 纯化 |
| 文章编号： | 1000-2286（2005）05-0753-06 |
| 收稿时间： | 2005-08-05 |
| 修稿时间： | 2005年8月5日 |
Construction of pMSL As a New Vector for Overexpressing Recombinant Proteins in Mycobacteria |
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| YE Zi-jian,LIU Chong,CHEN Xiao-you,CHANG Zeng-yi.Construction of pMSL As a New Vector for Overexpressing Recombinant Proteins in Mycobacteria[J].Acta Agriculturae Universitis Jiangxiensis,2005,27(5):753-758. |
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| Authors: | YE Zi-jian LIU Chong CHEN Xiao-you CHANG Zeng-yi |
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| Institution: | 1. The Department of Biology and Chemistry, Pingxiang College, Pingxiang 337000, China;2. The De partment of Biological Science and Biotechnology,Tsinghua University, Beijing 100084, China;3. Department of Bacriological Immunology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149; China ;4. College of Life Science, Peking University, Beijing 100871, China |
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| Abstract: | A novel Escherichia coli-Mycobacterium shuttle vector pMSL was constructed for the purpose of utilizing Mycobacterium smegmatis to express and purify Mycobacterium tuberculosis proteins.The vector contains the following: the promoter region of the hsp60 gene from M.bovis,a cloning site for inserting the target genes,an enhanced green fluorescence protein(EGFP) for monitoring the expression and the localization of target proteins,a thrombin cleavage site integrated between cloning site and EGFP gene for the removal of EGFP from purified fusion proteins,and the codons encoding histidine tag(His6) fused at the end of EGFP gene.The results indicated that the M.smegmatis transformed with pMSL could express EGFP efficiently.The EGFP protein thus expressed was purified effectively using the nickel resins via affinity chromatography. |
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| Keywords: | Mycobacterium tuberculosis plasmid pMSL EGFP overexpressing purification |
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