Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen

Background: Previously, we have identified several Per a 1 (CrPII) allergen s from a lambda gt22A cDNA library of Periplaneta americana. This study aim ed to sequence clone C42 and determine its molecular and antigenic properti es. Methods: The cDNA of C42 was sequenced and ligated into a bacteria expressi on vector, pET21. The recombinant proteins were purified by ion-exchange an d affinity chromatographies. Their antigenicities were analyzed by immunobl otting, ELISA, and binding inhibition with human IgE. Results: The nucleotide of the cDNA has been sequenced and the deduced amin o acid which encodes a 446-amino-acid protein (50kDa) determined. The recom binant C42 protein can bind both anti-Per a 1 monoclonal antibodies and hum an IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Seque nce homology searches revealed a high degree of identity to two other membe rs of the Per a 1 family, C17 and C6, and the German cockroach (Blattella g ermanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeals, and the crude B. germanica extract, Per a 1, and recombin ant allergens share similar antigenic determinant(s) as defined by ELISA an d IgE-binding inhibition studies. In IgE-binding epitope studies, an immuno positive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzy me fragments in E. coil. The shortest peptide, C42-P560, identified by mono clonal antibodies and human specific IgE, can inhibit IgE binding to C42. Conclusions: An additional Per a 1 allergen has been defined at the molecul ar level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, wh ich is an internal repeat. The results defined the boundaries of the antige nic site and will facilitate further epitope-mapping studies.