Cloning, expression and purification of Ag85A-ESAT6 antigens of Mycobacterium tuberculosis as a fusion protein

Abstract

Aims and objectives: Secretory antigens of Ag85 complex and 6-kDa Early Secreted Antigenic Target (ESAT-6) play a role in cell wall synthesis and virulence of Mycobacterium tuberculosis (MTB), respectively. They could induce a potent immune response and showed protective effects in some studies in animal models. So, they are considered as potential candidate antigens in new vaccination strategies against MTB. The aim of this study includes cloning, expression and purification of recombinant Ag85A-ESAT6 as a fusion protein for further studies of vaccine designation. Materials and Methods: A synthetic DNA fragment composed of coding sequences of Ag85A and ESAT6 was cloned into the BamHI and EcoRI restriction site of pET28a plasmid. Then the recombinant plasmid was transformed to Escherichia coli BL21 (DE3) and expressed in optimum condition. The expressed protein was analyzed and confirmed by SDS–PAGE and western blotting using anti-His tag and monoclonal anti-ESAT6 antibodies. Protein purification was performed by Ni–NTA spin column under denaturing conditions. Results: The results of SDS–PAGE and western blotting showed that Ag85A-ESAT6 recombinant fusion protein was successfully cloned and expressed with a His tag in its N-terminal. Considering the expression of this protein as an inclusion body, its purification was done by denaturing protocol. Conclusion: The Ag85A-ESAT6 fusion protein expressed in this study can be combined with a suitable adjuvant to induce the cellular and humoral immune responses, and its protective effects against MTB could be evaluated in subsequent studies