Molecular cloning and expression of MPT63 gene of Mycobacterium tuberculosis

Abstract

The present investigation was undertaken to amplify, clone and express MPT63 gene of Mycobacterium tuberculosis to discern this secretary protein as a potent diagnostic and immunogenic antigen. Primer specific for MPT63 gene with restriction enzyme sites, viz BamHI and HindIII were designed. MPT63 gene was amplified using DNA from M. tuberculosis 198/94 (IVRI) strain, with designed primers by polymerase chain reaction (PCR). The 412 bp amplicon was purified and first cloned into pGEM-T vector. Positive clone was confirmed by colony PCR and restriction enzyme digestion. The pGEM-T released insert was ligated with BamHI and HindIII digested pET32b expression vector using T4 DNA ligase and transformed into Escherchia coli. Recombinant clones were selected by colony PCR and restriction enzyme digestion and induced with 1mM final concentration of Isopropylß- D-thiogalactopyranosidase (IPTG) for expression of the recombinant MPT63 protein. The expressed protein of 33 kDa was obtained during 2 h post induction. Western blot with Ni- NTA anti-histidine HRP conjugate and hyperimmune serum raised in rabbits confirmed the presence and purity of recombinant MPT63 protein by immuno reactivity at the unique 33 kDa region. Further confirmation of recombinant protein was done by dot blot and indirect ELISA using rabbit hyperimmune serum. The recombinant MPT63 protein was evaluated as a skin test antigen to produce delayed type hypersensitivity reaction in guinea pigs. Recombinant MPT63 protein produced skin erythema of 3 to 4 mm diameter in guinea pigs sensitized with killed M. tuberculosis and M. bovis culture. From the present study, it may be concluded that understanding of the immunogenic components of an infectious agent is essential through molecular characterization, in regard to the serological diagnosis, and the development of strategies for efficient immune protection and eradication of the disease.