Molecular cloning, recombinant expression and IgE-binding epitope of ω-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis
Molecular cloning, recombinant expression and IgE-binding epitope of ω-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis
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DOI：
10.1111/j.1742-4658.2005.04858.x
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发表时间：
2005-09-01
期刊：
影响因子：
5.4
通讯作者：
Morita, E
中科院分区：
文献类型：
--
作者：
Matsuo, H;Kohno, K;Morita, E
小麦ω-5醇溶蛋白已被确定为小麦依赖性运动诱导的过敏反应的主要过敏原。我们在该蛋白的一级序列中检测到7个IgE结合表位。在这项研究中，我们在三名小麦依赖性运动诱导过敏反应患者中新发现了四个额外的IgE结合表位序列：QQFHQQQ、QSPEQQQ、YQQYPQQ和QQPPQQ。确定重组过敏原的可用性将有利于食物过敏的诊断和治疗。然而，由于ω-5醇溶蛋白基因尚未被克隆，重组蛋白目前不可用。我们试图克隆ω-5麦醇溶蛋白基因，并产生用于体外诊断工具的同质重组蛋白。使用基于PCR的策略，我们从小麦基因组DNA中分离出两个全长omega-5醇溶蛋白基因，命名为omega-5和omega-5 b，并确定了核苷酸序列。由omega-5a编码的蛋白质预计为439个氨基酸长，计算质量为53 kDa; omega-5 b基因将编码393个氨基酸，但它含有两个终止密码子，表明omega-5 b是假基因。通过pET系统在大肠杆菌中表达ω-5a麦醇溶蛋白的C-末端半部分（178个氨基酸），包括所有11个IgE结合表位序列，并使用RP-HPLC纯化。Westernblot分析和斑点印迹抑制试验表明，重组蛋白具有与IgE结合的能力。我们的研究结果表明，重组蛋白可以是一个有用的工具，用于识别患者与小麦依赖运动诱导过敏反应在体外。
Wheat omega-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 omega-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the omega-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 omega-5 gliadin genes, designated omega-5 and omega-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by omega-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the omega-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that omega-5b is pseudogene. The C-terminal half (178 amino acids) of the omega-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 omega-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro.