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目的构建结核分枝杆菌glcB基因原核表达工程株,并进行表达、纯化。方法用PCR扩增结核分枝杆菌glcB基因,并克隆入pTA2质粒。测序正确后,再亚克隆入pET30a(+)质粒,构建pET30a(+):glcB重组体,转化宿主菌大肠埃希菌BL21(DE3)。结果结核分枝杆菌glcB基因工程株经0.4 mM异丙基硫代-β-D-半乳糖苷(IPTG)诱导后,表达出相对分子质量为92 kD重组蛋白。SDS-PAGE分析显示,IPTG诱导4 h重组蛋白的表达量最高。表达蛋白以包涵体形式存在于胞质中,表达量占全菌蛋白质的50%。经Ni-NTA柱纯化,获得纯度为90%的重组蛋白。结论成功地构建了结核分枝杆菌glcB基因工程表达株,并获得GlcB重组蛋白,为研究新型血清学诊断活动性结核病奠定了基础。
Objective To construct the prokaryotic expression strain of mycobacterium tuberculosis glcB and to express and purify it. Methods Mycobacterium tuberculosis glcB gene was amplified by PCR and cloned into pTA2 plasmid. After sequencing, the recombinant plasmid pET30a (+) was subcloned into plasmid pET30a (+) to construct recombinant plasmid pET30a (+): glcB. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). Results After induced by 0.4 mM isopropylthioglucoside (IPTG), the M. tuberculosis glcB gene was expressed as a recombinant protein with a molecular weight of 92 kD. SDS-PAGE analysis showed that IPTG induced the expression of recombinant protein at 4 h. The expressed protein exists in the cytoplasm in the form of inclusion bodies, and accounts for 50% of the total bacterial protein. Purification by Ni-NTA column, to obtain a purity of 90% of the recombinant protein. Conclusion The glcB genetically engineered strain of Mycobacterium tuberculosis was successfully constructed and GlcB recombinant protein was obtained, which laid the foundation for the study of novel serological diagnosis of active tuberculosis.