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

Ahyar Ahmad, Abdul Karim, Rugaiyah A. Arfah, Rosana Agus, Rusdina Bte Ladju, Najdah Hidayah, Muhammad N. Massi, Astutiati Nurhasanah, Harningsih Karim, Irda Handayani, Rizal Irfandi

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

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Keywords


CbiX; Cloning; Expression; Mycobacterium tuberculosis; Rv0259c; B-cell-epitope; T-cell-epitope.

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DOI: 10.28991/ESJ-2024-08-04-07

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Copyright (c) 2024 Ahyar Ahmad, Abdul Karim, Rugaiyah Arfah, Rosana Agus, Rusdina Bte Ladju, Najdah Hidayah, Muhammad Nasrum Massi, Astutiati Nurhasanah, Harningsih Karim, Irda Handayani, Rizal Irfandi