SEARCH

SEARCH BY CITATION

Keywords:

  • allergen;
  • hsp 70;
  • molecular cloning;
  • Penicillium citrinum

Summary

BackgroundPenicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. However, detailed studies on allergens of this ubiquitous Penicillium species are still lacking.

Objective For the characterization of allergens of this prevalent Penicillium species, molecular cloning and expression of the allergen genes of P. citrinum were performed in the present study.

Methods Molecular cloning of the allergen genes was performed by using a λUni-Zap XR cDNA library of P. citrinum and serum from an asthmatic patient. The cloned cDNA was excised from the phage vector as a recombinant pBluescript phagemid and sequenced. The cDNA of the IgE-binding clone was expressed as fusion protein with the gultathione-S-transferase. The corresponding natural allergen was analysed by absorption immunoblotting using monoclonal antibody and serum from asthmatic patient. The frequency of IgE-binding to the allergen cloned was analysed by dot immunoassay using recombinant allergen and by immunoblotting using the whole extract of P. citrinum.

Results In the screening of cDNA library of P. citrinum using serum from an asthmatic patient, IgE-binding cDNA clones designated SC4 and XL were obtained. The 5′-truncated, 0.7-kb and 1.7-kb cDNA inserts of clones SC4 and XL contained open reading frames of 163 and 503 amino acids, respectively. On alignment, the deduced amino acid sequences showed that 97 (60%) of the 163 amino acids and 376 (75%) of the 503 amino acids were identical to the corresponding amino acid sequence of the human heat shock protein in the hsp70 family. Both recombinant SC4 and XL showed positive SDS-PAGE-immunoblot reactivity to a monoclonal antibody MA3-006 against the human hsp 70 protein. For characterization of the corresponding natural allergen, immunoblotting reactivities of MA3–006 and IgE antibodies to the 70kDa component of P. citrinum have been shown to be disappeared after absorption of these antibodies with the recombinant SC4 protein. Sera from 14 (41%) of 34 Penicillium-allergic patients showed IgE-binding to the recombinant XL protein and the 70kDa component in the extract of P. citrinum.

Conclusion Results obtained suggest that hsp 70 is an allergen of P. citrinum and that clones SC4 and XL contain partial cDNAs of this allergen gene.