Molecular Construction and Expression Analysis of Rv3875 and Rv2873 from Mycobacterium tuberculosis as Novel Protein Biomarkers for Tuberculosis Immunodiagnostics.

BACKGROUND: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), continues to become a significant risk to world health despite the availability of Bacille Calmette-Guérin (BCG) vaccination. The limited efficacy of BCG in adults and the low sensitivity of conventional diagnostic methods highlight the urgent need for novel antigens to improve TB immunodiagnostics.

METHODS: This study constructs, clones, and expresses Rv3875 and Rv2873, encoding ESAT-6 and MPT83 proteins from Mtb H37Rv as potential subunit proteins. Genomic DNA was extracted, and target genes were amplified by polymerase chain reaction (PCR) using a primer pair containing BamHI and HindIII restriction sites. Immunoinformatics analysis using IEDB predicted T-cell epitopes of Rv3875 and Rv2873. SignalP, DeepTMHMM, VaxiJen, AllerTOP, and ToxinPred2 were used to assess localization, antigenicity, and safety profiles.

RESULTS: The amplicons (288 bp for Rv3875 and 663 bp for Rv2873) were successfully cloned into the pTrcHis A expression vector and transformed into Escherichia coli DH5α and BL21 strains. Colony PCR, restriction digestion, and sequencing assured the presence and integrity of the recombinant constructs, showing over 99% identity to reference sequences. Recombinant protein expression induced with IPTG yielded bands of ~11-12 kDa (Rv3875) and ~22 kDa (Rv2873) on SDS-PAGE. SignalP and DeepTMHMM identified ESAT-6 as secreted and MPT83 as a lipoprotein, whereas VaxiJen, AllerTOP, and ToxinPred confirmed both as antigenic, nonallergenic, and non-toxic.

CONCLUSION: These results show that Rv3875 and Rv2873 successfully cloned and expressed at the molecular level, and that they could be used as subunit proteins for TB immunodiagnostics.