凡纳滨对虾次要过敏原肌质钙结合蛋白的质谱鉴定及免疫交叉反应

Mass spectrometry identification and immune cross-reactivity of a minor shrimp allergen-sarcoplasmic calcium binding protein from Litopenaeus vannamei

目的:质谱鉴定凡纳滨对虾相对分子质量(Mr)为21 000的次要过敏原组分肌质钙结合蛋白(SCP),分析它与其他虾、蟹等甲壳类过敏原的免疫交叉反应,以阐明SCP可作为甲壳类食物过敏原检测、检验及脱敏的标准过敏原物质。方法:运用MALDI-TOF/TOF-MS鉴定凡纳滨对虾Mr为21000的过敏原组分,利用软件BLAST、ClustalW分析甲壳类食物中该蛋白的氨基酸序列同源性,同时用纯化的21 000过敏原免疫小鼠制备其特异性多克隆抗体,通过该抗体与其他8种甲壳类食物蛋白粗提液的Western blot法结果分析该过敏原的免疫交叉反应。结果:质谱鉴定结果显示,凡纳滨对虾21 000过敏原为肌质钙结合蛋白;氨基酸序列同源性分析显示其与斑节对虾、中国对虾、克氏原螯虾、细趾小龙虾、褐虾的SCP序列一致性为81%～100%;Western blot结果显示,针对SCP的特异性多克隆抗体与凡纳滨对虾、刀额新对虾、斑节对虾、口虾蛄、罗氏沼虾、克氏原螯虾、远海梭子蟹、锈斑鲟、中华绒螯蟹粗提液在SCP对应的21 000左右处均有反应条带。结论:凡纳滨对虾Mr为21 000的次要过敏原为肌质钙结合蛋白,其氨基酸序列与多种甲壳类具有很高的同源性以及较强的免疫交叉反应。

AIM： To identify sarcoplasmic calcium-binding protein（SCP） as a minor shrimp allergen by mass spectrometry,and to analyze the immune cross-reactivity among crustacean SCPs.METHODS： The Mr 21 000 allergen from Litopenaeus vannamei was identified by MALDI-TOF/TOF-MS.BLAST and ClustalW were used to compare amino acid sequence identity of the allergen among crustaceans.The puritifed Mr 21 000 allergen was injected subcutaneously in mice to produce the specific polyclonal antibodies to analyze immune cross-reactivity of the allergen with proteins from 8 other species of crustaceans by Western blotting.RESULTS： The Mr 21 000 shrimp allergen was identified as SCP.Sequence comparison revealed that SCP had 81%-100% amino acid identity among crustaceans.Western blotting showed that the proteins with Mr about 21 000,corresponding to SCP from Metapenaeus ensis,Penaeus monodon,Oratosquilla oratoria,Macrobrachium rosenbergii,Procambarus clarkii,Portunus pelagicus,Charybdis feriatus,Eriocheir sinensis were recognized by polyclonal antibodies against SCP of Litopenaeus vannamei.CONCLUSION： SCP is a minor shrimp allergen,and SCPs have a high sequence homology and strong immune cross-reactivity among crustaceans,which can be used as detective,diagnostic and safe immunotherapeutic agents for subjects with shrimp allergy.