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

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Presentation transcript:

Molecular cloning of a major Alternaria alternata allergen, rAlt a 2 Robert K. Bush, MDa,b, Hiram Sanchez, BSa, David Geisler, AASa Journal of Allergy and Clinical Immunology Volume 104, Issue 3, Pages 665-671 (September 1999) DOI: 10.1016/S0091-6749(99)70340-4 Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 1 cDNA and deduced amino acid sequence for rAlt a 2. Nucleotide sequence (5′ to 3′) is for the Pribnow box, start codon, open reading frame, and stop codon. Deduced amino acid is in the open reading frame for the allergen. Stop codon TGA is denoted by an asterisk . Only the gene-encoding sequence is shown. Journal of Allergy and Clinical Immunology 1999 104, 665-671DOI: (10.1016/S0091-6749(99)70340-4) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 2 SDS-PAGE stained with Coomassie blue stain; molecular weight (MW) markers in kilodaltons. Lane 1, Crude A alternata allergen; lane 2, P pastoris media containing rAlt a 2; lane 3, affinity-purified rAlt a 2; lane 4, affinity-purified native Alt a 2. Journal of Allergy and Clinical Immunology 1999 104, 665-671DOI: (10.1016/S0091-6749(99)70340-4) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 3 Western Blot of affinity-purified rAlt a 2 (strips 1) and native Alt a 2 (strips 2) probed with murine mAbs to rAlt a 2. A to D represent mAbs used (A, 6A11F5; B, 10F2F8; C, 8A2F12; D, 9E9F2); J represents inhibition of mAb 10F2F8 binding to rAlt a 2 and native Alt a 2 by crude A alternata extract (see text). MW, Molecular weight markers; N, negative control (no first antibody). Journal of Allergy and Clinical Immunology 1999 104, 665-671DOI: (10.1016/S0091-6749(99)70340-4) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 4 Dot-blot autoradiograph of 4 samples: 1, crude A alternata extract; 2, nonrecombinant control P pastoris media; 3, rAlt a 2; 4, BSA control. Blots were incubated with pooled sera from subjects sensitive to A alternata sera (+) or pooled control sera from nonallergic subjects (–) . IgE binding was then detected by iodine 125–labeled rabbit anti-human IgE antibodies and autoradiography. All protein solutions were prepared at a concentration of 1 mg/mL. One hundred microliters was added to each well (dot). Journal of Allergy and Clinical Immunology 1999 104, 665-671DOI: (10.1016/S0091-6749(99)70340-4) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 5 Dot-blot autoradiographs showing IgE binding of 26 individual human sera to A alternata proteins: A, rAlt a 2; B, BSA (irrelevant protein); C, crude A alternata extract; strip N, incubated with sera of non-A alternata –sensitive individual. All protein solutions were prepared at a concentration of 0.1 mg/mL. One hundred microliters was added to each well (dot). Journal of Allergy and Clinical Immunology 1999 104, 665-671DOI: (10.1016/S0091-6749(99)70340-4) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 6 One percent agarose gel showing the amplified rAlt a 2 sequence in different A alternata strains. DNA markers, pGem DNA markers. A alternata ATCC strains: lane 1, 11680; lane 2, 188699; lane 3, A1002; lane 4, 46582; lane 5, no template; lane 6, no primers; lane 7, blank. Journal of Allergy and Clinical Immunology 1999 104, 665-671DOI: (10.1016/S0091-6749(99)70340-4) Copyright © 1999 Mosby, Inc. Terms and Conditions