ANALYSIS OF ALLERGENS IN BUCKWHEAT SEED STORAGE PROTEINS

从授粉后14天收获的荞麦（Fagopyrum esculenturn Moench）未成熟种子中分离到FA02和FA18基因，发现它们编码在种子发育过程中表达的豆科蛋白样蛋白。FA02的氨基酸序列与BW24KD的n端氨基酸结构域相同，BW24KD被认为是荞麦的主要过敏原。预测FA02将被裂解成两个独立的组分，一个41.3 kDa的α亚基和一个21 kDa的β亚基。从分子质量和N端氨基酸推测BW24KD是FA02的β -递质。对推断的FA02 β亚基进行了抗血清的制备，并对荞麦种子（Fagopyrum esculentum M.和Fagopyrum tartaricum M.）的总蛋白进行了免疫印迹分析，结果显示有多组蛋白与抗血清发生反应。根据在50%的过敏患者中与IgE发生强烈反应的10 kDa荞麦过敏原蛋白（BW10KD）的n端氨基酸序列，合成简并引物并进行3′race PCR扩增。从未成熟荞麦种子cDNA文库中分离到Fe2SA1 cDNA克隆，该克隆作为探针a3' RACE PCR克隆，与2S白蛋白种子贮藏蛋白部分相同。Fe2SA1的氨基酸序列与16和18 kDa分子部分相同，这两个分子被认为是主要的荞麦过敏原。在大肠杆菌中。在表达系统中，编码Fe2SA1的cDNA翻译产物被过敏患者血清强烈识别。

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.