Identification of the antigenic determinants of the American cockroach allergen Per a 1 by error-prone PCR.
The group I allergen of cockroach is found in both American and German cockroaches, designated as Per a 1 and Bla g 1, respectively. Members of these allergens so far identified are composed of tandem repeats that may cause the high allergenicity of Per a 1 allergen. In this study, we used monoclonal antibodies HW-8 and HW-19, which can inhibit the binding of patient IgE to Per a 1 allergen, to define the structure of the antigenic determinants in Per a 1.0103 (designated C3), an isoallergen of Per a 1 allergen. Two recognition sites are present, one in the N-terminus (aa 1-208) and the other in the C-terminus (aa 208-395). The N-terminal epitope is not accessible to antibody molecules on the pET-expressed C3 protein. The C-terminal epitope was further localized to the aa 267-354 region (C3E) by colony immunoscreening of the cDNA epitope library. By negative screening of the mutated C3E expression library generated by error-prone PCR (ER-PCR), an approach which has rarely been applied in epitope mapping, the functional epitope was identified to lie in aa 318-337 with aa 323-331 being the core motif. The minimal region of the functional epitope was further delineated, by sequence alignment, to be D-x-[I, L]-A-[I, L]-L-P-V-D-E-[L, I]-x-A-[L, I], where x represents any amino acids. This motif is found in all Per a 1 allergens and may serve as a basis for designing a peptide vaccine for allergen-specific immunotherapy. To our knowledge, this is the first report for (1) detailed mapping of the cockroach allergens and (2) use of error-prone PCR random mutagenesis and negative selection in molecular allergology.