Cloning and expression of Aspergillus fumigatus allergen Asp f 16 mediating both humoral and cell-mediated immunity in allergic bronchopulmonary aspergillosis (ABPA).
BACKGROUND
Aspergillus fumigatus, a ubiquitous fungus, is responsible for a number of lung disorders in atopic and non-atopic individuals. Standardized, pure, and relevant allergens are desirable for reliable immunodiagnosis of the disease and to understand the structural and functional properties of these allergens and the role they play in causing ABPA.


OBJECTIVE
Molecular cloning and characterization of a relevant allergen from A. fumigatus cDNA library.


METHODS
A cDNA library was constructed from 96 h old mycelium of A. fumigatus using lambda ZAP expression vector. A novel gene encoding an A. fumigatus allergen was identified by screening the library with sera from ABPA patients. The gene was cloned and the allergen over-expressed in Escherichia coli. This recombinant allergen, Asp f 16, was evaluated in ELISA and Western blots using sera from patients and normal subjects and peripheral blood mononuclear cells (PBMC) for antigen-induced stimulation.


RESULTS
Seventy percent of the patients with ABPA demonstrated high levels of serum IgE antibodies to Asp f 16, a 43-kDa protein, whereas patients with allergic asthma, Aspergillus skin test-positive asthmatics without clinical evidence of ABPA, and normal controls failed to show Asp f 16-specific IgE binding by ELISA. The deduced amino acid sequences of Asp f 16 showed extensive sequence homology to 30.6-kDa Asp f 9 at the N-terminal region of the protein. PBMC from the majority of patients with ABPA exhibited significant proliferation with the recombinant Asp f 16 allergen.


CONCLUSIONS
Specific humoral and cell-mediated immune responses of Af-sensitized patients against Asp f 16 suggest its usefulness in the immunodiagnosis of hypersensitivity diseases due to Af and understanding the pathophysiology of ABPA.
Banerjee
B
B
Department of Paediatrics, Allergy-Immunology Division, Medical College of Wisconsin, Milwaukee, WI 53295, USA.
Kurup
V P
VP
Greenberger
P A
PA
Johnson
B D
BD
Fink
J N
JN
D000485:Allergens / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000917:Antibody Formation; D000918:Antibody Specificity; D000946:Antigens, Fungal / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology*; D052179:Antigens, Plant; D001229:Aspergillosis, Allergic Bronchopulmonary / Q000209:etiology* / Q000276:immunology*; D001232:Aspergillus fumigatus / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005656:Fungal Proteins; D006801:Humans; D007111:Immunity, Cellular; D007114:Immunization; D007073:Immunoglobulin E / Q000378:metabolism; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D012882:Skin Tests
D000485:Allergens; D000946:Antigens, Fungal; D052179:Antigens, Plant; D005656:Fungal Proteins; C438315:allergen Asp f 16; D007073:Immunoglobulin E