ANALYSIS OF ALLERGENS IN BUCKWHEAT SEED STORAGE PROTEINS

荞麦(Fagopyrum Esculenturn Motch)基因FA02和FA18是从授粉14天后收获的未成熟种子中分离出来的，它们编码在种子发育过程中表达的豆蛋白。推导的氨基酸序列与BW24KD的N端氨基酸序列完全一致，BW24KD被认为是荞麦的主要过敏原。据预测，FA02将被切割成两个独立的组分，一个是41.3 kDa的α亚基，一个是21 kDa的β亚基。根据分子质量和N端氨基酸推测，BW24KD为FA02的β-亚基。以荞麦种子(Fagopyrum esculentum M.和Fagopyrum tartaricum M.)的总蛋白为模板，制备了FA02β亚基的免疫印迹抗血清。发现有几组蛋白质与抗血清发生反应。根据50%过敏患者中10 kDa荞麦过敏蛋白(BW10KD)与IgE强反应的N端氨基酸序列，设计简并引物，进行3‘RACE扩增。以A3‘RACE克隆为探针，从荞麦未成熟种子的cDNA文库中分离得到的Fe2SA1基因与2S白蛋白种子贮藏蛋白部分同源。Fe2SA1的氨基酸序列与16和18 kDa分子部分相同，认为是荞麦的主要过敏原。在大肠杆菌中。表达系统是编码Fe2SA1基因的翻译产物，能被过敏患者的血清强烈识别。

Buckwheat (Fagopyrum esculenturn Moench) genes FA02 and FA18 were isolated from immature seeds harvested 14 days after polination, and were found to encode legumin-like proteins that are expressed during seed development. The deduced amino acid sequence of FA02 was identical to the N-terminal amino acid domain of BW24KD, which is believed to be a major buckwheat allergen. It was predicted that FA02 would be cleaved to generate two separate components, a 41.3 kDa alpha-subunit and a 21 kDa beta-subunit. It was deduced from molecular mass and N terminal amino acid that BW24KD was the beta-submit of FA02. Antiserum was raised against the deduced FA02 beta-subunit and immunoblotting of total protein from buckwheat seeds (Fagopyrum esculentum M. and Fagopyrum tartaricum M.) revealed that several groups of proteins reacted with the antiserum. Based on the N-terminal amino acid sequence of the 10 kDa buckwheat allergenic protein (BW10KD) which strongly reacted with IgE in 50% of allergic patients, a degenerate primer were synthesized and 3'RACE PCR amplification was carried out. Fe2SA1 cDNA clone, which was isolated from a cDNA library made of immature buckwheat seeds using as a probe a3' RACE PCR clone, was partially identical to 2S albumin seed storage protein. The deduced amino acid sequence of Fe2SA1 was partially identical to the 16 and 18 kDa molecule, which are believed to be major buckwheat allergens. In an E. coli. Expression system, the translation product of a cDNA encoding the Fe2SA1 was strongly recognized by sera from allergic patients.