Molecular cloning of a major Alternaria alternata allergen, rAlt a 2

Background: Sensitivity to the fungus Alternaria alternata is a common cause of asthma, Epidemiologic studies from a variety of locations worldwide indicate that A alternata sensitivity is closely linked with the development of asthma. Furthermore, A alternata sensitivity has been associated with severe and potentially fatal attacks of asthma, Objective: The diagnosis of A alternata sensitivity is hampered by the lack of standardized and well-characterized allergenic extracts. Molecular cloning of allergens offers the possibility of providing large quantities of purified, well-characterized allergens not only for diagnostic purposes but also for studying the pathogenesis of A alternata sensitivity, We used molecular cloning to identify, purify, and produce a majors alternata allergen in quantity. Methods: We prepared messenger (m)RNA from A alternata to produce a complementary (c)DNA library The library was screened for A alternata allergens by using sera from A alternata-sensitive individuals. A recombinant allergen was isolated, the cDNA sequence was determined, and the protein was expressed in Pichia pastoris, Results: A unique A alternata allergen, rAlt a 2, was identified. A 2.2-kb cDNA sequence was obtained that has homology with a common transposable region and mouse RNA-dependent eukaryote initiation factor-2 a-kinase but no homology to any known allergen, No N-glycosylation sites were found in the cDNA sequence. The recombinant allergen was recognized by IgE antibodies in the sera of 16 of 26 (61%) individuals allergic to A alternata, which defines Alt a 2 as a major allergen. Conclusions: We have molecularly cloned a unique major A alternata allergen, rAlt a 2. Identification and expression of the recombinant allergen should enhance the development of standardized A alternata allergenic extracts and provide stable reagents for investigating the pathogenesis of A alternata sensitivity.