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

Background: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to underst and the allergenic composition of this ubiquitous fungal species. Objective: The complementary DNA (cDNA) clone of an allergen from P citrinu m was isolated and expressed in Escherichia coli as a fusion protein. mAbs were prepared with the recombinant protein as antigen. The corresponding na tural allergen in the fungal extracts was identified with the mAbs. Methods: A Uni-Zap XR P citrinum cDNA library was screened with sera from a sthmatic 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 BA LB/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 sam ples 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 acid s open reading frame. The deduced amino acid sequence of the encoded protei n has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxi somal membrane protein allergen (Asp f 3), PCES was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Pe nicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3 . In addition, 11 of the 13 Pen c 3-positive serum samples have IgE immunob lot 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. Fo ur of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The pr esence 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 mAb s 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 cDN A clone encoding this allergen. In addition, the mAbs generated may be usef ul in standardizing the diagnostic allergenic extracts.