Tsuji H, Hiemori M, Kimoto M, Yamashita H, Kobatake R, Adachi M, Fukuda T, Bando N, Okita M, Utsumi S
Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Juboki 111, Soja 719-1197, Japan.
Biochim Biophys Acta. 2001 Mar 19;1518(1-2):178-82. doi: 10.1016/s0167-4781(00)00310-9.
A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.
已分离出一个编码大豆过敏原Gly m Bd 28K的cDNA克隆。该克隆有一个1567 bp的cDNA插入片段，带有一个1419 bp的开放阅读框和一个148 bp的3'非翻译区，后面跟着一个聚腺苷酸化尾。开放阅读框被证明编码一个由473个氨基酸组成的多肽。从过敏原获得的肽段（包括其N端肽段）的化学测定氨基酸序列显示包含在从cDNA推导的氨基酸序列的N端区域，这表明cDNA的前半部分编码带有21个氨基酸前导片段的过敏原。包含过敏原的肽片段在大肠杆菌中作为与谷胱甘肽S-转移酶的融合蛋白表达，并与大豆敏感患者的血清和针对该过敏原的单克隆抗体进行免疫印迹分析。此外，同源性分析表明，该cDNA的多肽与南瓜种子中的MP27/MP32蛋白和胡萝卜球蛋白样蛋白具有高度同源性。这一发现表明，该多肽可能由作为信号肽和前体蛋白一部分的21个氨基酸片段组成，在大豆子叶发育过程中可能会转化为两种成熟蛋白，即Gly m Bd 28K和一种23 kDa的蛋白。
