Construction of the fundamental data for the clinical test of the allergy patients' substituted foods

过敏患者替代食品临床试验基础数据的构建

• 批准号: 12680128
• 负责人: ODANI Sumiko
• 金额: $ 2.11万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680128/
• 关键词: allergy; substituted food; hen egg; Japanese quail egg; ovalbumin; ovomucoid; antigenicity; epitope; Inhibition ELISA; 代替食品; 官能検査; 卵アレルギー; ウズラ卵白; 消化; ペプチド; 二次元電気泳動; イムノブロット

Ovalbumin (OVA) and ovomucoid (OM) are major allergens of hen's egg white and often induce hypersensitivity in atopic children. The sequence homology of Japanese quail (JQ) egg OVA^<1)> or OM^<2)> and hen (H) egg OVA^<3)> or OM^<4)> is 88.9% or 75.8% respectively. But the binding activities of JQ-OVA to rabbit anti H-OVA serum and JQ-OM to anti H-OM serum, measured by the method of inhibition ELISA, were 6.8% and 0.04% respectively. These results indicate that the antigenicity decreases dramatically when a few amino acid residues replace each other. This study aimed to characterize the antigenic determinants of JQ-OVA by immunoblotting and microsequence analysis methods. For this purpose, JQ-OVA was isolated from the JQ egg white by the fractionation with ammonium sulfate and gel filtration chromatography. The rabbit anti JQ-OVA serum was prepared by injecting the purified JQ-OVA to a female rabbit. JQ-OVA was digested with S. aureus V8 protease, TPCK-trypsin, TLCK-chymotrypsin and pepsin, and the peptides were separated with tricine SDS-polyacrylamide gel electrophoresis and two-dimentional gel electrophoresis. The antigenicity was tested by binding an anti JQ-OVA rabbit IgG antibody with the peptides. It is proposed that a part of the antigenic determinant (or epitope) existed in the JQ-OVA sequences of 17-28, 72-84, 1 20-124, 132-149, 160-175, 206-211, 219-226, 233-242, 244-253 and 298-308. These epitopes differed from those of H-OVA. Furthermore, baby foods (chawan-musi, custard pudding egg bouro, and kakitama soup) were cooked with JQ-egg and H-egg. A sensory test revealed that there were no differences between these baby foods cooked with JQ-egg and H-egg. Those results will allow better treatment to be provided for the hen egg allergy.1)Nisbet, et al. 1981 Eur. J. Biochem, 115, 335-345, 2)Kato, et al. 1987 Biochemistry, 26, 193-2013)Mucha,et al. 1990 Nucleic Acids Res., 18, 5553. 4)Papamakos, et al. 1982 J. Ml. Biol., 158, 515-537

卵清蛋白（OVA）和卵类粘蛋白（OM）是鸡蛋白色的主要过敏原，常引起特应性儿童过敏。日本鹌鹑卵（JQ）OVA^<1）>和OM^<2）>与母鸡卵（H）OVA^<3）>和OM^<4）>的同源性分别为88.9%和75.8%。用抑制ELISA法测得JQ-OVA与兔抗H-OVA血清的结合活性为6.8%，JQ-OM与兔抗H-OM血清的结合活性为0.04%。这些结果表明，当少数氨基酸残基相互替换时，抗原性显著降低。本研究旨在通过免疫印迹和微序列分析方法来表征JQ-OVA的抗原决定簇。采用硫酸铵分级分离和凝胶过滤层析从鸡卵白色中分离出鸡卵清蛋白。通过将纯化的JQ-OVA注射给雌性家兔制备兔抗JQ-OVA血清。JQ-OVA用S.金黄色葡萄球菌V8蛋白酶、TPCK-胰蛋白酶、TLCK-胰凝乳蛋白酶和胃蛋白酶，用Tricine SDS-聚丙烯酰胺凝胶电泳和双向凝胶电泳对肽段进行分离。通过将抗JQ-OVA兔IgG抗体与肽结合来测试抗原性。推测JQ-OVA序列17-28、72-84、120 -124、132-149、160-175、206-211、219-226、233-242、244-253和298-308中存在部分抗原决定簇（或表位）。这些表位不同于H-OVA的表位。此外，婴儿食品（chawan-musi，奶油布丁蛋布罗，和kakitama汤）与JQ-鸡蛋和H-鸡蛋煮熟。感官测试表明，这些婴儿食品之间没有区别与JQ鸡蛋和H鸡蛋煮熟。这些结果将允许更好地治疗鸡蛋过敏。1）Nisbet等人，1981 Eur. J. Biochem，115，335-345，2）Kato等人，1987 Biochemistry，26，193-2013）Mucha等人，1990 Nucleic Acids Res.，18，5553。4）Papamakos等人，1982 J. Ml.生物学：158，515-537