title: Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies.
abstract: The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein.
authors.affiliation: Institute of Biochemistry, College of Medicine, National Taiwan University, No. 1, Jen-AI Rd, Taipei, Taiwan. lupin@ha.mc.ntu.edu.tw
authors.forename: L P
authors.initials: LP
authors.lastname: Chow
authors.forename: S L
authors.initials: SL
authors.lastname: Liu
authors.forename: C J
authors.initials: CJ
authors.lastname: Yu
authors.forename: H K
authors.initials: HK
authors.lastname: Liao
authors.forename: J J
authors.initials: JJ
authors.lastname: Tsai
authors.forename: T K
authors.initials: TK
authors.lastname: Tang
mesh_terms: D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001232:Aspergillus fumigatus / Q000276:immunology*; D018604:Epitope Mapping; D005656:Fungal Proteins / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D011817:Rabbits
chemical_list: D000485:Allergens; D005656:Fungal Proteins; D007073:Immunoglobulin E