Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3)

Abstract

Background: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to understand the allergenic composition of this ubiquitous fungal species. Objective: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein, mAbs were prepared with the recombinant protein as antigen. The corresponding natural allergen in the fungal extracts was identified with the mAbs. Methods: A Uni- Zap XR P citrinum cDNA library was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein. The frequency of IgE binding to the expressed protein was analyzed by immunoblotting. Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation. Results: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients. An IgE-binding cDNA clone was obtained and designated as PCE2. PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame. The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3). PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Penicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3. In addition. 11 of the 13 Pen c 3-positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3. The presence of IgE cross- reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition. Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients. Conclusion: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum. PCE2 is a full-length cDNA clone encoding this allergen. In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts.