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

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 expression plasmid pMAL;p2;Ag85B successfully expressed MBP/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.