南极磷虾原肌球蛋白纯化鉴定、理化特性及模拟表位肽预测  

Purification,Physicochemical Properties,and Mimotope Peptide Prediction of Tropomyosin from Antarctic Krill(Euphausia superba)

作　　者：王珊 刘瑶 刘柯欣 张书琪 林松毅 孙娜[1,2,3] WANG Shan;LIU Yao;LIU Kexin;ZHANG Shuqi;LIN Songyi;SUN Na(School of Food Science and Technology,Dalian Polytechnic University,Dalian 116034,China;National Engineering Research Center of Seafood,Dalian 116034,China;Collaborative Innovation Center of Seafood Deep Processing,Dalian 116034,China)

机构地区：[1]大连工业大学食品学院,辽宁大连116034 [2]国家海洋食品工程技术研究中心,辽宁大连116034 [3]海洋食品精深加工关键技术省部共建协同创新中心,辽宁大连116034

出　　处：《食品科学》2022年第24期117-128,共12页Food Science

基　　金：国家自然科学基金优秀青年科学基金项目(32022067)。

摘　　要：对南极磷虾原肌球蛋白(tropomyosin,TM)进行提取纯化与质谱鉴定,并对其热稳定性、pH值稳定性及消化稳定性进行研究,同时利用生物信息学对其进行序列同源性分析、空间结构同源建模及过敏原模拟表位肽识别。十二烷基硫酸钠聚丙烯酰氨凝胶电泳结果显示,经蛋白粗提、热除杂、等电点沉淀、硫酸铵盐析等多步提纯,最终仅在32 kDa附近得到一条清晰条带。利用超高效液相色谱-质谱联用对其进行扫描,经UniProt数据库检索确定该目的蛋白为TM(Euphausia superba),蛋白分子质量32.6 kDa,肽段覆盖率达97%。同源性分析结果表明南极磷虾TM是一种高度保守蛋白,与26种甲壳动物的TM序列同源性在88%~98.2%之间。南极磷虾TM对热、酸碱及胃液消化均具有较强的稳定性,而对肠液消化稳定性较差,易被胰蛋白酶和糜蛋白酶降解生成低分子质量肽段。通过DNAStar Protean、ANTHEPROT、BepiPred 1.0 server、ABCpred server、Immunomedicine Group 5种生物信息学工具最终预测识别出8条南极磷虾TM模拟表位肽(EAQNKETNAKADKADDEVH、DLERSEERLN、TKLAEASQAADESER、EADRKYDE、ERAEERAEAG、VSEEKANQREEAYKEQI、RSVQKLQKEVDR、VNEKEKYKGI),并在其空间结构中进行一一映射。本研究为南极磷虾TM模拟表位肽的精准预测识别及其相关低致敏性产品的开发提供了科学依据。Tropomyosin(TM) from Antarctic krill(Euphausia superba) was extracted,purified,and identified by mass spectrometry,and its thermal stability,pH stability and digestive stability were studied.Meanwhile,sequence homology analysis,spatial structure homology modeling,and allergen mimotope peptide recognition were carried out using bioinformatics.The crude protein was extracted,thermally treated to remove impurities,precipitated at the isoelectric point and purified by salting-out.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) of the purified protein showed only one clear band with molecular mass near 32 kDa.The target protein was identified as TM(Euphausia superba)by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) analysis and UniProt database search.The molecular mass of the protein was 32.6 k Da and the peptide coverage was 97%.The results of homology analysis and physicochemical properties showed that TM from Antarctic krill was a highly conserved protein,and its sequence homology with those from 26 crustacean species ranged from 88% to 98.2%.TM from Antarctic krill had strong stability to heat,acid,alkali and gastric juice digestion,but poor stability to intestinal juice digestion,and was easily degraded by trypsin and chymotrypsin to produce low-molecular-mass peptides.Eight mimotope peptides(EAQNKETNAKADKADDEVH,DLERSEERLN,TKLAEASQAADESER,EADRKYDE,ERAEERAEAG,VSEEKANQREEAYKEQI,RSVQKLQKEVDR,VNEKEKYKGI) in Antarctic krill TM were finally predicted and identified by five bioinformatics tools including DNAStar Protean,ANTHEPROT,BepiPred 1.0 server,ABCpred server,and Immunomedicine Group,and mapped in the spatial structure.This study provides a scientific basis for the accurate prediction and identification of mimotope peptides in TM from Antarctic krill and the development of hypoallergenic products.

关 键 词：南极磷虾 原肌球蛋白 提取纯化 蛋白鉴定 稳定性 生物信息学 模拟表位肽 

分 类 号：TS202.1[轻工技术与工程—食品科学]