CLONING AND B-CELL-EPITOPE MAPPING OF MPT64 FROM MYCOBACTERIUM-TUBERCULOSIS H37RV

The gene of the immunogenic protein MPT64 found in culture filtrates o f Mycobacterium tuberculosis H37Rv was cloned and sequenced. A compari son showed mpt64 and the gene encoding MPB64 from Mycobacterium bovis BCG Tokyo to be identical except for one silent mutation. The regions encoding the promoter and the signal peptide were also well conserved for the two sequences. Southern blot experiments on genomic mycobacter ial DNA shelved the presence of mpt64 in the M. tuberculosis substrain s H37Rv, H37Ra, and Erdman and in the M. bovis BCG substrains Tokyo, M oreau, and Russian, whereas the M. bovis BCG substrains Glaxo, Pasteur , Canadian, Tice, and Danish 1331 and Mycobacterium leprae lack the ge ne. Southern blot analyses revealed differences in the restriction enz yme patterns within the M. tuberculosis substrains as well as within t he M. bovis BCG substrains, indicating either different chromosomal lo calization of mpt64 or that mutations have occurred at different locat ions on the chromosomes. N-terminal and C-terminal deletion mutants we re constructed for the mapping of B-cell epitopes on MPT64 with five m onoclonal antibodies, C24b1, C24b2, C24b3, L24b4, and L24b5. Western b lot (immunoblot) analysis revealed that the murine antibodies bind to one linear and three conformational epitopes.