characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen. Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen Key words: American cockroach; cross-reactive allergen; cDNA cloning; IgE-binding epitope; Per a 1 (Cr-PII) allergen. Background: Previously, we have identi®ed several Per a 1 (Cr-PII) allergens from a lgt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties. Methods: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were puri®ed by ion-exchange and af®nity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE. Results: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50 kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as de®ned by ELISA and IgE-binding inhibition studies. In IgEbinding epitope studies, an immunopositive C42 fragment was ®rst identi®ed from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identi®ed by monoclonal antibodies and human speci®c IgE, can inhibit IgE binding to C42.Conclusions: An additional Per a 1 allergen has been de®ned at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358±446 of C42, which is an internal repeat.The results de®ned the boundaries of the antigenic site and will facilitate further epitope-mapping studies.
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