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

在过敏性患者中，许多患者因摄入谷物而引起过敏。小麦是世界上产量和消费量最大的粮食作物。为了阐明小麦的过敏原性，我们用过敏性皮炎患者的血清筛选小麦中的过敏原，发现约15种过敏原。其中，17-kDa的过敏性蛋白（Tri a Bd 17 K）已被鉴定为α-淀粉酶抑制剂CM 16，其具有天冬酰胺连接的糖链。在本研究中，我们试图纯化Tri a Bd 27 K，一种主要的小麦过敏原，并阐明其性质。通过10%NaCl溶液提取、硫酸铵分级沉淀、Q-Sepharose层析、层析聚焦和苯基-Sepharose层析等方法从小麦粉中纯化了Tra Bd 27 K。纯化的蛋白对PAS试验呈阳性反应，表明该过敏原是N-Asn连接的糖蛋白。凝集素染色显示该糖蛋白含有α-L-岩藻糖和α-D-甘露糖。N-末端氨基酸序列分析表明，该过敏原是一种未知蛋白。根据Tri a Bd 27 K及其肽段的N-末端氨基酸序列，合成简并寡聚DNA作为PCR引物，以小麦子叶发育过程中的poly（A）^+RNA为模板，通过PCR扩增，获得了Tri a Bd 27 K及其肽段的DNA片段。采用RT-PCR方法获得适合于cDNA克隆的探针，PCR产物经鉴定为编码变应原肽段的片段，并以此为探针进行进一步的实验。

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.