不同方法提取和处理中华绒螯蟹组织蛋白的过敏原组分分析  

Analysis of allergic components in tissue protein of eriocheir sinensis with different extraction and proocessing methods

作　　者：朱黎娜 侯丽英[1] 张盈莹 周宇捷[1] 李韶深[2] 李会强[1] 

机构地区：[1]天津医科大学医学检验学院,天津300203 [2]天津市中医药研究院附属医院,天津300120

出　　处：《中国免疫学杂志》2014年第12期1652-1657,共6页Chinese Journal of Immunology

基　　金：国家自然基金项目资助项目(81371882)

摘　　要：目的:分析不同方法提取和处理中华绒螯蟹组织的蛋白组分及过敏原组分,为检测中华绒螯蟹特异性IgE提供最佳抗原制备方法。方法:分别采用PBS提取法、丙酮抽提法和裂解液提取法提取中华绒螯蟹组织蛋白,并用裂解液提取法分别提取加热前、后的蟹蛋白;采用SDS-PAGE和双向电泳分析蛋白组分,采用Western blot分析过敏原组分。结果:不同方法提取的蛋白组分、sIgE与蛋白结合的强度和所识别的蛋白组分存在一定差异。PBS提取液蛋白主要分布在94、85、76、66、18kD,丙酮提取液蛋白主要分布在94、85、76、36 kD,裂解液提取液蛋白主要分布在200、125、51、43、38 kD。PBS提取液与sIgE结合的阳性反应条带主要分布在76、66、53、43、38、18 kD;丙酮提取液主要分布在94、76、66、50、43、38、18 kD;裂解液提取液主要分布在200、105、94、76、43、38、18 kD。加热前、后蟹蛋白过敏原组分差别不大。结论:裂解液提取法提取的中华绒螯蟹过敏原更适于制备测定特异性IgE的抗原。加热不影响蟹过敏原组分和与特异性IgE的结合活性。Objective：To analyze protein and allergic components in Eriocheir sinensis tissue with different extraction and processing methods , provide an optimal antigen extraction protocol for detecting specific IgE of Eriocheir sinensis.Methods： The proteins were extracted from tissue protein of Eriocheir sinensis with PBS extraction method ,acetone extraction method ,lysate extraction method,respectively;Extract heating and without heating tissue protein of Eriocheir sinensis with lysate extraction method .The total protein components were analyzed by SDS-PAGE and two-dimensional electrophoresis.The allergen components were identified by Western blot.Results：The binding strength of protein components extracted by different methods and sIgE in the immunoblot assay dif -fered.Protein components extracted by PBS extraction method were mainly 94,85,76,66,18 kD,by acetone extraction method were mainly 94,85,76,36 kD,while by lysate extraction method were mainly 200,125,51,43,38 kD.Protein components extracted by PBS extraction method reacted with patient serum were mainly 76,66,53,43,38 and 18 kD,by acetone extraction method were mainly 76, 66,53,43,38 and 18 kD,while by lysate extraction method are mainly 200,105,94,76,43,38 and 18 kD.Heating and without heating proteins and allergic components were similar.Conclusion：Lysate extraction method is more suitable for determination of specific IgE antigen preparation purpose.The binding activity of sIgE and heating or without heating allergen components is similar .

关 键 词：中华绒螯蟹 食物过敏 过敏原制备 加热 免疫活性 

分 类 号：R392.8[医药卫生—免疫学]