CLONING AND EXPRESSION OF MPT83 PLUS MPT64 FUSION PROTEIN FROM Mycobacterium tuberculosis IN Escherichia coli BL21 (DE3) STRAIN AS VACCINE CANDIDATE OF TUBERCULOSIS

Abstract

Mycobacterium tuberculosis drug-resistant strains have emerged, creating a new difficulty in the treatment of tuberculosis around the world and prompting the World Health Organization to declare TB an international emergency. With TB treatment resistance increasing, developing more potent vaccinations can ensure the TB epidemic is stopped or reduced. The MPT64 gene was amplified from the genomic DNA of the M. tuberculosis local strain using polymerase chain reaction (PCR) in vitro and then transferred into the pGEM-T Easy-Mpt83+Mpt64 vector and expressed in the E. coli BL21 (DE3) strain. Plasmid DNA was isolated and amplified using PCR, followed by DNA sequencing. Subcloned into the expression vector pTrcHisA on NheI/HindIII cloning site before being converted into the E. coli BL21 (DE3) strain were the correct recombinant MPT83 and MPT64 gene fusions. The white recombinant colony was propagated and maintained using 40 µM IPTG, then cells and protein recombinants were harvested, and SDS-PAGE electrophoresis was used to identify it. The protein was then purified with a 6XHis tagged-agarose beads purification kit. The results showed that the MPT83 and MPT64 gene fusion was successfully produced, expressed, and purified. The fusion protein of MPT83 and MPT64 with a His tag from the expression vector had a molecular weight of about 46 kDa and was expressed as a soluble protein. Cloned from the host strain of the E. coli BL21 (DE3) strain cell, the fusion genes were successfully produced in the bacteria. Pathways to tuberculosis diagnoses are laid by the purified recombinant fusion proteins, MPT83 and MPT64, and may be more successful in the future as a vaccine candidate.