Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene

A genomic DNA region, designated RD1, that is present in virulent and clini cal strains of Mycobacterium tuberculosis and M. bovis, has been shown to b e deleted in bacillus Calmette Guerin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF-10, ORF-13 and ORF-15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosi s genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX-4T ve ctor system and expressed in Escherichia coli as fusion proteins with gluta thione-S-transferase (GST). The recombinant proteins appeared as major cell ular proteins in SDS-PAGE gels at the expected molecular mass, The identity of each fusion protein was confirmed by reactivity with anti-GST antibodie s in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST-ORF-14 fusion prot ein reacted in Western immunoblots. The protein corresponding to ORF-13 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 prot ease. In Western immunoblots, the purified ORF-13 protein reacted with anti bodies in 26 of 57 human sera (46%) from TB patients while no reactivity wa s seen with Il sera from M. bovis BCG-vaccinated healthy subjects. Interest ingly, sera from nine of 15 (60%) long-term contacts of TB patients also ha d 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.