Screening of food allergy-causing factors and study on wheat allergy.

食物过敏因素筛查及小麦过敏研究.

基本信息

批准号：
11680138
负责人：
KIMOTO Masumi
金额：
$ 2.37万
依托单位：
Okayama Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680138/
关键词：
Wheat Allergen Tri a Bd 27K glycoprotein cDNA IgE cDNA cloning Tri A Bd 27K sugar protein allergenicity

项目摘要

Of allergic patients, many patients have been suffering from allergy eliciting by ingestion of cereals. Wheat is the most important foodstuff of the cereals in view of its yield and consumption quantity in the world. In order to elucidate the allergenicity of wheat, we screened the allergens in wheat using the sera of wheat-sensitive patients with atopic dermatitis and found about 15 allergens. Among them, a 17-kDa allergic protein (Tri a Bd 17K) had been identified as α-amylase inhibitor CM 16 with an Asn-linked sugar chain. In the present study, we attempted to purify Tri a Bd 27K, a major wheat allergen, and to elucidate its properties. Furthermore, we investigated the cloning of cDNA encoding the allergen to reveal information concerning its complete primary structure.Tri a Bd 27K was purified to near homogeneity from wheat flour by extraction with 10 % NaCl, ammonium sulfate fractionation, Q-Sepharose chromatography, chromatofocusing and phenyl-Sepharose chromatography. The purified protein showed positive response to the PAS test, indicating that the allergen is N-Asn-linked glycoprotein. The glycoprotein was shown to have α-L-fucose and α-D-mannose by the specific lectin staining. N-Terminal amino acid sequencing showed that the allergen is an unkown protein. Further, the allergen was digested with trypsin in gel and two peptides were purified by HPLC.On the basis of the N-terminal amino accid sequences of the Tri a Bd 27K and its peptides, some degenerate oligoDNAs were synthesized to use as primers of PCR.Using poly (A)^+RNA prepared from developing wheat cotyledons, RT-PCR was done to obtain a probe suitable for the cloning of cDNA.Since the PCR products were confirmed to encode the peptide fragments of the allergen, the products were used as probes in the further experiments.

在过敏患者中，许多患者因摄入谷物而引起过敏。鉴于小麦的产量和消费量，小麦是世界上最重要的谷物食品。为了阐明小麦的致敏性，我们对小麦进行了筛选。利用小麦敏感的特应性皮炎患者的血清对小麦中的过敏原进行了研究，发现了大约 15 种过敏原，其中有一种 17-kDa 的过敏蛋白（Tri a. Bd 17K）已被鉴定为具有 Asn 连接糖链的 α-淀粉酶抑制剂 CM 16 在本研究中，我们尝试纯化主要的小麦过敏原 Tri a Bd 27K，并阐明其特性。克隆编码过敏原的 cDNA，以揭示有关其完整一级结构的信息。通过用 10% 的提取物从小麦粉中提取 Tri a Bd 27K，将其纯化至接近均质NaCl、硫酸铵分级、Q-Sepharose 层析、层析聚焦和苯基-Sepharose 层析对PAS 测试显示阳性反应，表明过敏原是N-Asn 连接的糖蛋白，显示具有α-L。 -岩藻糖和α-D-甘露糖通过特异性凝集素染色显示，该过敏原是一种过敏原。进一步，用凝胶中的胰蛋白酶消化过敏原，并通过HPLC纯化两个肽。在Tri a Bd 27K及其肽的N端氨基酸序列的基础上，合成了一些简并的寡DNA以用作。 PCR引物。使用从小麦发育子叶中制备的poly(A)^+RNA，对适合克隆cDNA的探针进行RT-PCR。由此获得PCR产物被证实编码过敏原的肽片段，产物被用作进一步实验的探针。