tailieunhanh - Báo cáo khoa học: Molecular cloning, recombinant expression and IgE-binding epitope of x-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis

Wheatx-5 gliadin has been identified as a major allergen in wheat-depend-ent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exer-cise-induced anaphylaxis in this study. | ềFEBS Journal Molecular cloning recombinant expression and IgE-binding epitope of x-5 gliadin a major allergen in wheat-dependent exercise-induced anaphylaxis Hiroaki Matsuo Kunie Kohno and Eishin Morita Department of Dermatology Shimane University Schoolof Medicine Izumo Japan Keywords wheat allergy gliadin allergen recombinant Correspondence H. Matsuo Department of Dermatology Shimane University Schoolof Medicine 89-1 Enya-cho Izumo Shimane 693-8501 Japan Fax 81 853 21 8317 Tel 81 853 20 2210 E-mail hmatsuo@ Received 30 May 2005 accepted 12 July 2005 doi Wheat x-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences QQFHQQQ QSPEQQQ YQQYPQQ and QQPPQQ in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However because m-5 gliadin gene has not been cloned recombinant protein is currently unavailable. We sought to clone the m-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length m-5 gliadin genes designated m-5 and m-5b from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by x-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa the x-5b gene would encode a 393 amino acid but it contains two stop codons indicating that x-5b is pseudogene. The C-terminal half 178 amino acids of the x-5a gliadin protein including all 11 IgE-binding epitope sequences was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native x-5 gliadin purified from