结核分支杆菌Ag85B融合蛋白表达载体的构建及纯化

Construction and purification of antigen 85B fusion protein expression vector of mycobacterium tuberculosis

目的:构建结核分支杆菌Ag85B基因在大肠杆菌的融合表达载体,制备MBP/Ag85B融合蛋白并将其纯化。方法:常规方法构建重组表达质粒pMAL-p2-Ag85B,内切酶EcoRⅠ/HindⅢ酶切鉴定;重组表达质粒pMAL-p2-Ag85B转化大肠杆菌,经0.3mmol/L异丙基-β-D-硫代半乳糖苷(isopropyl-β-Dthiogalactoside熏IPTG)诱导表达产生融合蛋白(MBP/Ag85B),Westernblot鉴定表达产物。pMAL纯化树脂(Amyloseresin)对MBP/Ag85B融合蛋白纯化。结果:重组质粒pMAL-p2-Ag85B经EcoRⅠ/HindⅢ酶切后,1%琼脂糖凝胶电泳证实Ag85B正向插入pMAL-p2载体,重组菌经IPTG诱导后,有约70kD蛋白表达,与预期的融合蛋白分子量相符,蛋白表达量约占细菌总蛋白量的40%;Westernblot显示,MBP/Ag85B融合蛋白与人抗结核IgG抗体呈特异性反应;经Amyloseresin纯化后,MBP/Ag85B融合蛋白的纯度可达95%以上。结论:成功构建结核分支杆菌Ag85B重组融合蛋白表达质粒并制备及纯化其融合蛋白,为进一步研究Ag85B生物学功能及其抗原表位分析奠定了物质基础。

Objective： To construct the antigen 85B fusion protein expression vector of mycobacterium tuberculosis （MT） in Escherichia coli（E.coli） and prepare specific antibody to Ag85B. Methods： Ag85B gene was separated from pUC18-Ag85B and then inserted into the downstream of malE of expression vector pMAL-p2. The recombinant expression plasmid pMAL-p2-Ag85B was converted into E,coli,and the MBP/Ag85B fusion protein was expressed by induction of Isopropyl-β-D thiogalactoside （IPTG）. The recombinant fusion protein was identified by Western blot test and purified with Amylose resin after sonication. Results： Endonucleases digestion confirmed that Ag85B gene was inserted into the prokaryotic expression vector pMAL-p2. The recombinant exDression Dlasmid pMAL-p2-Ag85B successfully expressed MRP/Ag85B fusion protein in E.coli, and MBP/Ag85B fusion protein showed specific immunological reaction with anti-MT antibody. The purity of the MBP/Ag85B fusion protein was over 95% after purification with Amylose resin. Conclusion： The construction of the prokaryotic expression plamsid of Ag85B and the preparation of MBP/Ag85B fusion protein establish a solid basis for further studying the biological function of Ag85B and preparing the specific antibody against Ag85B.