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|標題:||Identification of the antigenic determinants of the American cockroach allergen Per a 1 by error-prone PCR|
|期刊/報告no：:||Journal of Immunological Methods, Volume 276, Issue 1-2, Page(s) 163-174.|
|摘要:||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 I 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 0), an isoallergen of Per a I 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-DE-[L, I]-x-A-[L, I], where x represents any amino acids. This motif is found in all Per a I 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. (C) 2003 Elsevier Science B.V All rights reserved.|
|Appears in Collections:||分子生物學研究所|
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