结核分枝杆菌glcB基因原核表达载体的构建、表达和纯化

Construction and Expression of the Prokaryotic Expression Vector of Mycobacterium tuberculosisglcB Gene and Its Purification

目的构建结核分枝杆glcB基因原核表达载体，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。结果以重组体转化BL21（DE3）后，经0．4mM异丙基硫代-β．D．半乳糖苷（IPTG）诱导，表达出分子量为92KD重组蛋白。SDS—PAGE分析显示，IPTG诱导4h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。结论成功地构建原核表达载体pET30a（＋）：glcB，并获得GIcB重组蛋白，为血清学诊断活动性结核病奠定了基础。

Objective To construct prokaryotic expression vector carrying glcB gene from Mycobacterium tuberculosis and express it in E.. coil Methods The m.tb glcB gene was amplified by PCR and then cloned into plasmid pTA2. After sequencing , the gene was cloned into plasmid pET30a（＋） to construct recombinant prokaryotic expression vector pET30a（＋）： gIcB. Results After transformation of the E.. coil and induction with 0.4 mmol/L of IPTG, recombinant target protein GIcB with Mr 92KD was expressed in pET3Oa（＋）：glcB system, and the expressed protein was maximum when induced with IPTG for 4 h; The expressed protein existed in cytoplasm in unsoluble form and amounted to 50% of total protein of E. coil The purity of the protein puttied through the Ni-NTA resin reached 90%. Conclusion The prokaryotic expression vector pET30a（＋）： gIcB was constructed successfully and the recombinant protein GIcB was obtained , which laid the foundation for application of the diagnose of active tuberculosis.