Epitope mapping of the Mycobacterium bovis secretory protein MPB70 using overlapping peptide analysis

The gene encoding the major Mycobacterium bovis secreted protein MPB70 was cloned and isolated from a DNA library in lambda EMBL 3, and the restriction map of the area of chromosome containing the gene ascertained. After sub-cloning, the complete DNA sequence and predicted amino acid sequence were determined, and from this information a series of overlapping octapeptides encoding all possible linear epitopes of eight or less amino acids were synthesized. These peptides were probed with monoclonal antibodies specific for M. bovis and with sera from M. bovis-infected cattle. Epitopes defined by this technique were then examined using a substitution analysis that allowed the significance of each amino acid in the putative epitope to be quantified, and the exact specificity of the antibody response for the epitope determined.