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|標題:||Cloning of the American cockroach Cr-PII allergens: Evidence for the existence of cross-reactive allergens between species||作者:||Wu, C.H.
|關鍵字:||American cockroach;Cr-PII allergen;cDNA cloning;cross-reactive;allergen;german blattella-germanica;monoclonal-antibodies;periplaneta-americana;bronchial-asthma;major allergens;fecal;extracts;whole-body;hypersensitivity;identification;purification||Project:||Journal of Allergy and Clinical Immunology||期刊/報告no：:||Journal of Allergy and Clinical Immunology, Volume 101, Issue 6, Page(s) 832-840.||摘要:||
Background: previously, we have identified the 28 and 32 kd proteins as additional important allergens from the American cockroach (Periplaneta americana) Cr PII allergenic fraction. Objective: The aim of this study was the cloning of P. americana Cr-PII allergens. Methods: A lambda gt22A cDNA library constructed from P. americana mRNA was packaged into Escherichia call Y1090 (r(-)), and clones recognized by murine anti-Cr-PII monoclonal antibodies and human IgE antibodies were isolated, sequenced, and subcloned into pET 21 and expressed in E. call BL21(DE3). Results: Six Cr-PII-positive clones recognized by human IgE antibodies were isolated. Two clones, C6 and C17, were sequenced, and we found 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 sequences with a 94% identity between them. C6 and C17 showed 59% and 77.3% skin reactivities, respectively, on 22 cockroach-sensitive atopic patients. Both clones were found to have 28.9% to 31,8% identities to ANG12 protein, a precursor of the African malaria mosquito (Anopheles gambiae) and 82.7% to 85.1% identity to a nucleotide sequence of the German cockroach (Blattella germanica) Bla g Bd90K allergen. The anti-C6 and anti-C17 antibodies were able to recognize Cr PII, recombinant proteins, five commercial American extracts, and two German cockroach extracts. Moreover, the binding of anti-C6 and anti C17 antibodies to recombinant protein can be inhibited by B. germanica crude extract. Furthermore, 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 great aid in facilitating the epitope mapping and improving diagnostic and therapeutic reagents for both cockroach species.
|Appears in Collections:||分子生物學研究所|
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