MOLECULAR-CLONING AND EXPRESSION OF A PENICILLIUM-CITRINUM ALLERGEN WITH SEQUENCE HOMOLOGY AND ANTIGENIC CROSS-REACTIVITY TO A HSP-70 HUMANHEAT-SHOCK-PROTEIN

Background Penicillium citrinum has been identified as the most preval ent 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 prev alent Penicillium species, molecular cloning and expression of the all ergen genes of P. citrinum were performed in the present study. Method s Molecular cloning of the allergen genes was Fel-formed by using a la mbda Uni-Zap XR cDNA library of P. citrinum and serum from an asthmati c patient. The cloned cDNA was excised from the phage vector as a reco mbinant pBluescript phagemid and sequenced. The cDNA of the IgE-bindin g clone was expressed as fusion protein with the gultathione-S-transfe rase. The corresponding natural allergen was analysed by absorption im munoblotting using monoclonal antibody and serum from asthmatic patien t. The frequency of IgE-binding to the allergen cloned was analysed by dot immunoassay using recombinant allergen and by immunoblotting usin g the whole extract of P. citrinum. Results In the screening of cDNA l ibrary of P. citrinum using serum from an asthmatic patient. IgE-bindi ng 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 rea ding frames of 163 and 503 amino acids. respectively. On alignment, th e deduced amino acid sequences showed that 97 (60%) of the 163 amino a cids and 376 (75%) of the 503 amino acids were identical to the corres ponding amino acid sequence of the human heat shock protein in the hsp 70 family. Both recombinant SC4 and XL showed positive SDS-PAGE-immuno blot reactivity to a monoclonal antibody MA3-006 against the human hsp 70 protein, Far characterization of the corresponding natural allerge n. immunoblotting reactivities of MA3-006 and IgE antibodies to the 70 kDa 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 t o the recombinant XL protein and the 70 kDa component in the extract o f P. citrinum. Conclusion Results obtained suggest that hsp 70 is an a llergen of P. citrinum and that clones SC4 and XL contain partial cDNA s of this allergen gene.