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

在过敏患者中，许多患者因摄入谷物引起过敏。从产量和消费量来看，小麦是世界上最重要的粮食。为了阐明小麦的致敏性，我们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筛选，发现约15种过敏原。其中，一个17 kda的过敏蛋白（Tri a b17k）被鉴定为α-淀粉酶抑制剂CM 16，其糖链为asn链。在本研究中，我们试图纯化Tri a b27k，一个主要的小麦过敏原，并阐明其性质。此外，我们研究了编码过敏原的cDNA的克隆，以揭示其完整的初级结构信息。采用10% NaCl萃取、硫酸铵分馏、Q-Sepharose层析、聚焦层析和苯基sepharose层析等方法，从小麦粉中纯化出Tri a b27k。纯化后的蛋白对PAS试验反应阳性，表明该变应原为N-Asn-linked糖蛋白。凝集素特异性染色表明该糖蛋白具有α-L-聚焦和α- d -甘露糖。n端氨基酸测序结果显示该过敏原为未知蛋白。用胰酶消化凝胶，用高效液相色谱法纯化两个肽段。以Tri a bd27k及其肽的n端氨基酸序列为基础，合成了一些退化寡核苷酸作为PCR引物。利用小麦发育子叶中制备的poly (A)^+RNA，采用RT-PCR方法获得了适合于cDNA克隆的探针。由于PCR产物被证实编码了过敏原的肽片段，因此将其作为进一步实验的探针。

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.