R K Bush1, H Sanchez, D Geisler. 1. William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin 53705, USA.
Abstract
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 major A 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 alpha-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.
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 major A 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 alpha-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.
Authors: Päivi M Salo; Ming Yin; Samuel J Arbes; Richard D Cohn; Michelle Sever; Michael Muilenberg; Harriet A Burge; Stephanie J London; Darryl C Zeldin Journal: J Allergy Clin Immunol Date: 2005-09 Impact factor: 10.793