中文摘要：

以日本鳗鲡皮与肌肉组织为研究对象,采用碱溶、酸溶、盐析、冻干等方法纯化得到胶原蛋白,采用加热、饱和硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析等方法纯化得到两种亚型的小清蛋白（PV-Ⅰ和PV-Ⅱ）,纯化的目标蛋白经动物特异性抗体的免疫印迹实验确证。酶联免疫吸附测定和免疫印迹分析结果显示,纯化的胶原蛋白和小清蛋白分别与鱼类过敏患者阳性血清发生特异性反应,且二者之间无免疫交叉反应。体外模拟胃液消化实验和SDS-PAGE分析结果显示,胶原蛋白和小清蛋白均具有较高的消化稳定性。结果提示,日本鳗胶原蛋白和小清蛋白具有较高的消化稳定性和免疫原性,二者可引发不同患者的IgE介导特异性超敏反应。

英文摘要：

As the largest producer and consumer of freshwater fish in the world,many people suffer from allergy for consuming freshwater fish in China. However,the allergen profiles of freshwater fish are rarely known. Parvalbumin（PV） and collagen represent the major allergens involved in IgE-mediated food hypersensitivity to fish and fish products. A variety of clinical symptoms,such as urticaria,angioedema,respiratory symptoms（asthma and rhinoconjunctivitis） ,gastrointestinal symptoms（diarrhea and vomiting） and,in severe cases,fatal anaphylaxis can be caused after ingestion of fish. In this paper,collagen was isolated from Japanese eel（Anguilla japonica） via alkali dissolving,acid dissolution,salt precipitation,freeze-drying;and two subtypes of parvalbumin（PV-Ⅰ,PV-Ⅱ） were purified from Japanese eel with the methods of boiling,fractionation with ammonium sulphate,and DEAE-Sepharose anion-exchange chromatography. Then collagen and parvalbumin were identified by Western-blot with rabbit anti-collagen polyclonal antibody and by Dot-blot with mouse anti-parvalbumin monoclonal antibody respectively. After that,collagen and PV-Ⅰ,PV-Ⅱ were detected the cross immunoreaction of IgE binding to the fish-allergenic sera with enzyme linked immunosorbent assay（ELISA） and Dot-blot,and there is no cross immunoreaction to the fish-allergenic sera among collagen and PV-Ⅰ,PV-Ⅱ. Finally,the digestibility of collagen and parvalbumin was analyzed in simulated gastric fluid（SGF） digestion system. And the results showed that collagen and parvalbumin have high digestive stability. In conclusion,our present results indicated that collagen and parvalbumin from Japanese eel have high digestive stability and immunogenicity,and they can induce the IgE-mediated food hypersensitivity of different patients.