cDNA cloning and immunologic characterization of a novel EF-1beta allergen from Penicillium citrinum.

ABSTRACT

BACKGROUND: We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma. METHODS: A lambda-Uni-ZAP XR-based cDNA library of P. citrinum was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and heterologously expressed in Escherichia coli. The frequency of IgE-binding to the expressed protein and the IgE reactivity to allergen subunits were analyzed by immunoblotting. RESULTS: An IgE-reactive cDNA clone (clone B) was isolated by plaque immunoassay. The cDNA insert is 876-bp long and encodes a 228-amino acid polypeptide with a calculated molecular mass of 25 035 Da. Protein database search with the deduced clone B sequence revealed that 121 (53%) and 82 (36%) of the 228 amino acids were identical to those of the elongation factor 1-beta (EF-1beta) proteins from the yeast Saccharomyces cerevisiae and the parasite Echinococcus granulosus, respectively. His-tagged recombinant clone B proteins were constructed and expressed in E. coli. Seven (8%) of the 92 serum samples from patients with bronchial asthma showed IgE-binding to the recombinant clone B protein. Among these seven positive sera, five demonstrated IgE-binding to the C-terminal fragment (aa 119-228) while the other two sera showed IgE reactivity to the N-terminal fragment (aa 1-118) of this newly identified EF-1betaPenicillium allergen. CONCLUSIONS: A novel P. citrinum allergen (Pen c 24) was identified and characterized in the present study. Results obtained provide more information about allergens of prevalent airborne fungi and a basis to understand more about the IgE responses in human atopic disorders and in parasitic infections.