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

Previously, we have identified several Per a 1 (Cr-PII) allergens from a deltagt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties.

The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE.

The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50kDa) 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 defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42.

An additional Per a 1 allergen has been defined 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 defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.