【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-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N i-NTA亲和层析柱很好地被纯化。更多还原

【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.更多还原