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.
Institute of Molecular Biology, National Chung Hsing University, 250 Kuo Kuang Rd., Taichung 402, Taiwan. cyyang@dragon.nchu.edu.tw
Wei-Jen
WJ
Tsai
Ching-Hang
CH
Liu
Shu-Tsung
ST
Chen
Chiou-Ying
CY
Yang
D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D052179:Antigens, Plant; D001666:Binding Sites, Antibody; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010526:Periplaneta / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D000939:Epitopes; D011994:Recombinant Proteins; C091463:allergen Per a I