摘要
目的:鱼类是主要的致敏食物之一。痕量的过敏原即可能导致过敏患者产生严重的过敏反应,因此,本研究拟对多种鱼类主要过敏原小清蛋白的存在情况进行分析及相对定量检测。方法:采用热抽提获得鱼肌肉中小清蛋白的粗提物,通过Tricine-SDS-PAGE和Western blot分析粗提物中蛋白的存在情况,利用ELISA法对不同鱼类白肉和红肉中的小清蛋白含量进行测定。结果:Tricine-SDS-PAGE和Western blot结果显示粗提物蛋白主要为小清蛋白,分子量在10～14kD并具有一定的种属特异性。在不同的鱼类中存在着不同数量的小清蛋白异构体,它们与抗蛙小清蛋白单克隆抗体PARV-19的反应程度存在差异,表明小清蛋白的含量有所不同,与PARV-19结合的表位也可能存在一定的差异。ELISA结果显示,不同鱼类白肉中小清蛋白的含量为红肉中的4～33倍。结论:本研究建立的方法可用于多种经济鱼类主要过敏原小清蛋白的有效检测。
Objective：To develop an indirect enzyme-linked immunosorbent assay （ELISA） for semi-quantification of major allergen parvalbumin in fish.Methods：The soluble proteins were prepared from both white and dark muscles of seven species of freshwater fish and five species of marine fish.Tricine-SDS-PAGE and Western blot were performed to examine the protein patterns of fish muscle extracts.Natural parvalbumin being used to make calibration curve was purified from silver carp （Hypophthalmichthy molitrix） by ammonium sulphate fractionation,followed by ion exchange and gel filtration chromatography.The molecular mass of purified protein was estimated by Tricine-SDS-PAGE and identified by Western blot with anti-frog parvalbumin monoclonal antibody PARV-19.ELISA using PARV-19 was carried out to evaluate parvalbumin contents in white and dark muscles.Results：Tricine-SDS-PAGE revealed species-specific differences in proteins of heated extracts.Western blot confirmed that the major bands were showed in Tricine-SDS-PAGE with the molecular masses of 10-14 kD corresponded to parvalbumins recognized by PARV-19 and various numbers of isoforms of parvalbumin existed in different species of fish.There might be some differences in the parvalbumin contents and the epitope region was recognized by PARV-19 based on the differences in relative intensities of protein immunodetection.The ELISA showed that the contents of parvalbumin were 4 to 33 folds higher in the white muscle than in the dark muscle and varied greatly in different species of fish.Conclusion：These results validate that the dark muscle might be less allergenic than the white muscle due to the lower content of parvalbumins,and it is suggested that the commercial anti-parvalbumin antibody PARV-19 can be used to detect parvalbumins from the commercially important species of fish tested in this study and the method we develope succeeds to detect the major allergen in various species of fish.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2010年第8期716-720,723,共6页
Chinese Journal of Immunology
基金
国家自然科学基金(20872049)
十一五国家科技支撑计划重大项目(2008BAD94B09)资助
