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

在过敏患者中，许多患者一直患有因摄入谷物而引起的过敏。从产量和消费量来看，小麦是世界上最重要的粮食作物。为了阐明小麦的过敏性，我们用小麦过敏性特应性皮炎患者的血清对小麦中的过敏原进行了筛选，发现了约15种过敏原。其中，一个17 kDa的过敏蛋白(Tri A Bd 17K)已被鉴定为带有天冬氨酸连接糖链的α-淀粉酶抑制因子CM16。在本研究中，我们试图提纯小麦的主要过敏原Tri a Bd 27K，并阐明其性质。此外，我们还对该变应原基因的克隆进行了研究，以揭示其完整的一级结构信息。通过10%的氯化钠提取、硫酸铵分级沉淀、Q-琼脂糖层析、层析和苯基-琼脂糖层析，从小麦面粉中纯化得到了接近均一的Tra Bd 27K。纯化的蛋白对PAS试验呈阳性反应，表明该变应原为N-天冬氨酸连接的糖蛋白。凝集素特异性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，该变应原是一种未知蛋白质。在此基础上，根据Tri a Bd 27K及其多肽的N端氨基酸序列，合成了一些简并的寡核苷酸，并以小麦子叶发育过程中制备的Poly(A)^+RNA为模板，进行RT-PCR，获得了一条适合于克隆该变应原的探针，证实了该变应原的多肽片段为编码该变应原的多肽片段，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.