Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis‐Specific RD1 Region Gene

Abstract

A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guérin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF‐10, ORF‐14 and ORF‐15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX‐4T vector system and expressed in Escherichia coli as fusion proteins with glutathione‐S‐transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS‐PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti‐GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST‐ORF‐14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF‐14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST‐ORF‐14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF‐14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG‐vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long‐term contacts of TB patients also had antibodies reactive to the ORF‐14 protein. These results suggest that the ORF‐14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis.

Cellular Immune Responses to Recombinant Mycobacterium bovis BCG Constructs Expressing Major Antigens of Region of Difference 1 of Mycobacterium tuberculosis

“…However, obtaining full-length purified RD1 proteins that are free of contaminants, either from cultures of M. tuberculosis or using recombinant expression and purification technologies, is technically demanding and quite cumbersome (31)(32)(33). Therefore, to overcome the problems associated with obtaining full-length proteins, pools of synthetic peptides corresponding to each RD1 ORF were used in this study.…”

Efficient Testing of Large Pools of Mycobacterium tuberculosis RD1 Peptides and Identification of Major Antigens and Immunodominant Peptides Recognized by Human Th1 Cells

“…When tested with human serum, ORF14 was found to be a major antigen recognized by antibodies in sera of TB patients (1). Interestingly, ORF14 was predicted only by Amoudy et al but not by others (7) ( Table 1), and several studies have confirmed that ORF14 is a real protein coding gene of M. tuberculosis that is expressed under both in vitro and in vivo growth conditions (1,8,11). These findings suggest that to identify additional antigens of diagnostic and/or vaccine relevance, a systematic screening of all RD1 ORF proteins should be performed for immunological reactivity.…”

Comparative Evaluation of MPT83 (Rv2873) for T Helper-1 Cell Reactivity and Identification of HLA-Promiscuous Peptides in Mycobacterium bovis BCG-Vaccinated Healthy Subjects

“…However, obtaining full-length proteins from cultures of pathogenic M. tuberculosis is extremely hazardous and technically demanding (39). On the other hand, the production of purified recombinant mycobacterial proteins has often been notoriously difficult in E. coli (1,2,12). To overcome the problems associated with the expression and purification of recombinant mycobacterial proteins, pools of overlapping synthetic peptides have been successfully used in the past to replace recombinant or natural M. tuberculosis proteins in Th1 cell assays (40,41,55).…”