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The Natural Expression of Genes Encoding Major Antigens of Rd1 and Rd9 in M. Tuberculosis and other Mycobacteria

Shumaila N. M. Hanif, Rajaa Al-Attiyah and Abu Salim Mustafa

The natural expression of genes of five major antigenic proteins present in the genomic region of difference (RD)1, i.e. pe35, ppeE68, esxA and esxB , and RD9, i.e. esxV , was determined, at mRNA and protein levels, in M. tuberculosis, M. bovis BCG, and environmental mycobacterial species of M.avium and M.vaccae . The expression of mRNA was determined using total mycobacterial RNA and gene-specific forward and reverse primers in reverse transcription-polymerase chain reaction (RT-PCR), and the expression of proteins was determined by Western immunoblot analysis of mycobacterial sonicates with anti-sera containing protein-specific antibodies raised in rabbits. Furthermore, the expression of each protein in various sub-cellular fractions of M.tuberculosis was studied by Western immunoblot analysis of cell walls, culture filtrates and sonicates of M.tuberculosis using the rabbit anti-sera. The results showed mRNA and protein expressions for PE35, PPE68, ESXA, and ESXB only in M. tuberculosis , whereas ESXV was expressed in M.tuberculosis and M.bovis BCG, but none of them were expressed in M.avium and M.vaccae . Furthermore, all of the proteins were detected in various sub-cellular fractions of M. tuberculosis. In conclusion, our study demonstrates that the five major antigenic proteins encoded by genes present in RD1 and RD9 regions are naturally expressed in M.tuberculosis , whereas only ESXV is expressed in M. bovis BCG and none of these proteins are expressed in the environmental mycobacterial species of M. avium and M. vaccae . As these proteins are major antigens recognized by Th1 cells of protective phenotype, they could be useful in specific diagnosis and/or development of new vaccines against tuberculosis.