Identification of the antigenic determinants of the American cockroach allergen Per a 1 by error-prone PCR
| Autor: | Shu-Tsung Chen, Ching-Hang Liu, Wei-Jen Tsai, Chiou-Ying Yang |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
medicine.drug_class
Molecular Sequence Data Immunology Monoclonal antibody Polymerase Chain Reaction Epitope Epitopes Tandem repeat immune system diseases Immunoscreening Complementary DNA medicine Animals Periplaneta Immunology and Allergy Amino Acid Sequence biology Linear epitope Antibodies Monoclonal Allergens Antigens Plant respiratory system Molecular biology Recombinant Proteins respiratory tract diseases Epitope mapping Mutagenesis biology.protein Binding Sites Antibody Antibody Sequence Alignment Epitope Mapping |
| Zdroj: | Journal of Immunological Methods. 276:163-174 |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/s0022-1759(03)00105-4 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect