南极磷虾主要过敏原的分离纯化及性质研究  

Isolation,purification and properties of Antarctic krill(Euphausia superba)major allergens

作　　者：林娜 马德蓉 刘志东[1] 倪玲 段蕊 张俊杰 LIN Na;MA Derong;LIU Zhidong;NI Ling;DUAN Rui;ZHANG Junjie(East China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Shanghai 200090,China;Jiangsu Ocean University,School of Food Science and Engineering,Jiangsu,Lianyungang 222005,China)

机构地区：[1]中国水产科学研究院东海水产研究所,上海200090 [2]江苏海洋大学食品科学与工程学院,江苏连云港222005

出　　处：《水产学报》2022年第3期445-456,共12页Journal of Fisheries of China

基　　金：国家自然科学基金青年科学基金(32001624)。

摘　　要：为了探究南极磷虾致敏问题,从南极磷虾中筛选、鉴定、分离纯化其主要过敏原,并对过敏原的性质进行研究,实验通过缓冲盐溶液提取南极磷虾蛋白;采用SDS-PAGE和Western blot(WB)筛选南极磷虾过敏原;使用高效液相色谱-串联质谱鉴定过敏蛋白;通过等电点沉淀、硫酸铵盐析、阴离子交换柱层析分离纯化过敏原;采用SDS-PAGE及WB分析南极磷虾主要过敏原的耐热性及对模拟胃肠液的消化稳定性。结果显示,南极磷虾肌浆蛋白(SP)和肌原纤维蛋白(MF)电泳条带丰富且分子量范围广。南极磷虾蛋白与虾蟹过敏患者血清能发生免疫反应,其中至少有4个蛋白条带发生了阳性反应;免疫反应最强烈的蛋白分子量约为35 ku,能够被所有的患者血清识别,经液质联用鉴定此过敏蛋白为南极磷虾原肌球蛋白(TM)。硫酸铵分级盐析纯化TM的最佳饱和度为50%;分离纯化后得到了纯度较高的TM,其等电点为4.4,热稳定性好,能与虾蟹过敏患者血清发生强烈的免疫反应。TM随着模拟胃液消化时间的延长,主条带分子量逐渐降低,最后稳定在33 ku左右,并且分子量为15和12 ku的降解片段含量逐渐增多且稳定存在;这些降解条带仍然能与过敏患者血清发生较强的免疫反应。随着模拟肠液消化时间的延长,TM原始片段逐渐减少直至消失,最后完全降解成分子量更小的多肽;TM经肠液消化后其降解产物免疫活性大大降低。研究表明,南极磷虾中存在过敏原,TM是其最主要的过敏原,且具有免疫反应性。TM还具备了过敏原的一般特性,比如耐高温、耐胃蛋白酶消化、对胰蛋白酶消化部分稳定。研究结果对于阐明南极磷虾潜在致敏问题的预警和防控具有现实意义,也为南极磷虾加工利用及过敏原消减技术构建提供基础信息。Antarctic krill(Euphausia superba)is a type of crustacean living in Antarctic waters and is an important strategic marine living resource.Currently,the research on the safety of Antarctic krill is mainly focused on fluorine and arsenic,while the study on Antarctic krill sensitization is rarely reported.How to prevent and control the food allergy caused by Antarctic krill has become urgent work.In order to explore the sensitization problem of Antarctic krill,the main allergens were screened,identified,isolated and purified from Antarctic krill,and the properties of major allergens were studied.Antarctic krill proteins were extracted by salt buffer;Allergens were screened by SDS-PAGE and Western blot(WB);The primary structure of these allergic proteins were identified using high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS);Major allergens were separated and purified by isoelectric precipitation,ammonium sulfate salting out and anion exchange column chromatography.The heat resistance and the digestive stability of Antarctic krill major allergens to simulated gastrointestinal fluid were analyzed by SDS-PAGE and WB.Results showed that the protein bands of sarcoplasmic protein(SP)and myofibrillar protein(MF)extracted from Antarctic krill were rich and the range of the molecular weight(MW)was wide.Antarctic krill proteins could react with the serum IgE of shrimps/crabs allergic patients,and at least four proteins had positive reactions.The protein with the strongest immune reaction had a MW of about 35 ku and could be recognized by the serum of all allergic patients.This allergic protein was identified as Antarctic krill tropomyosin(TM)by LC-MS/MS.The optimum saturation of ammonium sulfate was 50%for the salting out Antarctic krill TM.After separation and purification,TM with high purity was obtained,which could have a strong immunoreactivity with the serum IgE of shrimps/crabs allergic patients.The measured isoelectric point of Antarctic krill TM was 4.4,and it had good thermal stability.With

关 键 词：南极磷虾 过敏原 原肌球蛋白(TM) 分离纯化 免疫反应性 消化稳定性 

分 类 号：TS254[轻工技术与工程—水产品加工及贮藏工程]