Molecular Cloning and In silico Epitope Prediction of Culture Filtrate Protein 10 and Resuscitation-promoting Factor D from Mycobacterium tuberculosis with Potential Immunodiagnostic Applications

Abstract

BACKGROUND: Tuberculosis (TB) is regarded as one of the most challenging infectious diseases globally, posing significant obstacles in terms of diagnosis and treatment. The limited effectiveness of the Bacillus Calmette-Guérin vaccine and the low sensitivity of conventional diagnostic methods underscore the need for improved antigen-based detection strategies. This study aimed to clone, express, and characterize culture filtrate protein 10 (CFP-10) and resuscitation-promoting factor D (rpfD) and to predict potential T-cell and B-cell epitopes using in silico methods. METHODS: Genes encoding Rv3874 (CFP-10) and Rv2389c (rpfD) were amplified from Mycobacterium tuberculosis H37Rv, subsequently cloned into the pCold II vector and then expressed in Escherichia coli BL21 (DE3). Induction with isopropylthio-β-galactoside resulted in recombinant protein expression and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In silico analysis included physicochemical characterization, secretion and localization prediction, and identification of T-cell and B-cell epitopes, followed by screening for antigenicity, allergenicity, and toxicity. RESULTS: Polymerase chain reaction successfully produced fragments of 303 bp (Rv3874) and 465 bp (Rv2389c), and sequencing confirmed the correct construct. SDS-PAGE revealed recombinant proteins of ~11 kDa (CFP-10) and ~15-16 kDa (rpfD), consistent with theoretical sizes. Both proteins were predicted to be antigenic and extracellular. Multiple strong binding epitopes were identified with high antigenicity, which were nontoxic and nonallergenic which signal a large immunogenic capacity. CONCLUSIONS: CFP-10 and rpfD were successfully cloned and expressed and demonstrated promising immunogenic profiles in silico. These findings support the potential as complementary antigens in future vaccine platforms and immunodiagnostic tools for TB.

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