Cloning and characterization of an exported protein present in the rd7 region of clinical isolates of mycobacterium tuberculosis

Abstract

The bacterium Mycobacterium tuberculosis is responsible for causing the disease newlinetuberculosis in mammals, which is regarded as one of the oldest diseases haunting the human race. The only available tuberculosis vaccine Bacillus Calmette-Guerine (BCG), is effective against childhood tuberculosis but is regarded as having low efficacy in conferring protection in the case of tuberculosis in adults. A comparison of the M. tuberculosis H37Rv strain and clinical isolates from Kerala had earlier revealed that the clinical strains have a distinctive 4.5 kb genomic sequence that is lacking from the H37Rv strain in the RD7 region. The RD7 is a distinctive genomic region that is absent in M. tuberculosis H37Rv and Mycobacterium bovis BCG strain. The 4.5 kb genomic sequence is projected to include 6 potential ORFs by newlineNCBI ORF prediction tool, one of which Novel Hypothetical Protein (NHP2) is anticipated to encode an exported protein with a length of 268 amino acids. Studies demonstrate that Mycobacterium tuberculosis secretory proteins such as the Ag85 complex, the ESAT-6 family protein, and the PE-PPE family proteins were newlineeffective vaccine candidates because they trigger T cells. Here, we present an indepth analysis of the exported protein, which is 268 amino acids long. The putative exported protein with a gene 807 bp long was PCR amplified and cloned in the expression vector pET-32a for expression. The protein was over expressed using Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and was isolated and purified using column chromatography. Bioinformatics studies were conducted to study the characteristics of the expressed protein. A novel putative mycobacterial protein discovered by subtractive hybridization was studied for its potential as a vaccine candidate using cutting-edge computer technologies.