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.