CLONING OF THE AMERICAN COCKROACH CR-PII ALLERGENS - EVIDENCE FOR THEEXISTENCE OF CROSS-REACTIVE ALLERGENS BETWEEN SPECIES

Background: previously, we have identified the 28 and 32 kd proteins a s additional important allergens from the American cockroach (Periplan eta americana) Cr PII allergenic fraction. Objective: The aim of this study was the cloning of P. americana Cr-PII allergens. Methods: A lam bda gt22A cDNA library constructed from P. americana mRNA was packaged into Escherichia call Y1090 (r(-)), and clones recognized by murine a nti-Cr-PII monoclonal antibodies and human IgE antibodies were isolate d, sequenced, and subcloned into pET 21 and expressed in E. call BL21( DE3). Results: Six Cr-PII-positive clones recognized by human IgE anti bodies were isolated. Two clones, C6 and C17, were sequenced, and we f ound encoding proteins of 228 and 274 amino acids with no cysteine or any potential N-glycosylation site, with predicted masses of 25.8 and 31.14 kd, respectively. Both molecules contain internal repeated seque nces with a 94% identity between them. C6 and C17 showed 59% and 77.3% skin reactivities, respectively, on 22 cockroach-sensitive atopic pat ients. Both clones were found to have 28.9% to 31,8% identities to ANG 12 protein, a precursor of the African malaria mosquito (Anopheles gam biae) and 82.7% to 85.1% identity to a nucleotide sequence of the Germ an cockroach (Blattella germanica) Bla g Bd90K allergen. The anti-C6 a nd anti-C17 antibodies were able to recognize Cr PII, recombinant prot eins, five commercial American extracts, and two German cockroach extr acts. Moreover, the binding of anti-C6 and anti C17 antibodies to reco mbinant protein can be inhibited by B. germanica crude extract. Furthe rmore, Northern blot analyses have shown that B. germanica mRNAs could be detected by both cDNA probes. Conclusion: Our findings provide the first evidence of antigenic cross-reactivity between P. americana and B. germanica allergens on molecular levels. The results will be a gre at aid in facilitating the epitope mapping and improving diagnostic an d therapeutic reagents for both cockroach species.