Expression and Epitope Prediction of the Sirohydrochlorin Cobaltochelatase Isolated from a Local Strain of Mycobacterium Tuberculosis

Abstract

The efficacy of the BCG vaccine, a widely recognized tuberculosis vaccine, has shown varying degrees of effectiveness, ranging from 0% to 80%. Sirohydrochlorin cobaltochelatase (CbiX), found in Mycobacterium tuberculosis, plays a crucial role in the bacteria metabolism, making it a promising target for future vaccine and drug development. Although several studies had been published regarding its role in M. tuberculosis vitamin B-12 metabolism, the potentials of CbiX protein as a vaccine candidate had not been widely discussed nor explored. This study focuses on the cloning and expression of the Rv0259c gene obtained from a clinical isolate of M. tuberculosis, as well as the exploration of CbiX protein epitopes. The Rv0259c gene was isolated by PCR, cloned into the pGEM®-TEasy vector, and subsequently sub-cloned into the pTrcHisA expression vector. Sanger sequencing, followed by BLASTN and BLASTX analyses, confirmed the presence of the CbiX protein-encoding gene. The amino acid sequence was predicted using BioEdit v.7.0.11, and a three-dimensional (3D) model was generated using SwissModel. Exploration for both B and T-cell epitopes was conducted using IEDB Ellipro, MHCI, and MHCII tools, revealing highly immunogenic epitopes, indicating the potential of CbiX as a vaccine candidate. Alignment using MAFFT between the putative amino acid sequence and CbiX proteins available in the NCBI database identified an amino acid variation (A182), situated outside the B-cell epitopes but within the T-cell epitopes. In silicoanalysis of HLA allele frequency predicted vaccine coverage of 86.14%±10.77%, with two significant epitope cores identified: AASAHPHVT and RRVAVASFL (both highly antigenic and showing high-frequency HLA allele binding), suggesting the protein might be a potential antigen for a future vaccine candidate. Doi: 10.28991/ESJ-2024-08-04-07 Full Text: PDF

Keywords

• cbix
• cloning
• expression
• mycobacterium tuberculosis
• rv0259c
• b-cell-epitope
• t-cell-epitope.