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

卵白蛋白(OVA)和类卵粘蛋白(OM)是蛋清的主要过敏原，常导致特应性儿童过敏。序列同源性分别为88.9%和75.8%。抑制法测定JQ-OVA与兔抗H-OVA血清的结合活性为6.8%，JQ-OM与抗H-OM血清的结合活性为0.04%。这些结果表明，当几个氨基酸残基相互替代时，抗原性显著降低。本研究旨在通过免疫印迹和微序列分析的方法对JQ-OVA的抗原决定簇进行鉴定。为此，采用硫酸铵分级和凝胶过滤层析的方法从JQ蛋清中分离得到JQ-OVA。将纯化的JQ-OVA注射到雌性兔体内，制备兔抗JQ-OVA血清。JQ-OVA用金黄色葡萄球菌V8酶、TPCK-胰酶、TLCK-糜蛋白酶和胃蛋白酶消化，用Tricine SDS-聚丙烯酰胺凝胶电泳法和双向凝胶电泳法分离多肽。用抗JQ-OVA兔免疫球蛋白抗体与多肽结合检测其抗原性。推测JQ-OVA的17-28、72-84、120-124、132-149、160-175、206-211、219-226、233-242、244-253和298-308序列中存在部分抗原决定簇(或表位)。这些表位与H-OVA的表位不同。此外，婴儿食品(茶碗-木丝、奶油布丁鸡蛋Bouro和kakitama汤)是用JQ-鸡蛋和H-鸡蛋烹制的。感官测试显示，这些用JQ-鸡蛋和H-鸡蛋烹调的婴儿食品没有区别。这些结果将为鸡蛋过敏症提供更好的治疗方法。1981欧元。J.Biochem，115,335-345，2)加藤等人。1987年生物化学，26,193-2013)Mucha，et al.1990年《核酸研究》，18,5553。4)Papamakos等人。1982年J.M.生物，158,515-537