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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。
OBJECTIVE: To identify the minor allergen component sarcoplasmic reticulum calcium (SCP) protein of Litopenaeus vannamei with its relative molecular mass (Mr) of 21 000 by mass spectrometry, and to analyze its immune cross-reaction with other crustacean allergens such as shrimp and crab. To clarify that SCP can be used as a standard allergen for the detection, testing and desensitization of crustacean food allergens. Methods: MALDI-TOF / TOF-MS was used to identify the allergen component of Mr 21000 in shrimp Litopenaeus vannamei. The amino acid sequence homology of the protein in crustacean food was analyzed by software BLAST and ClustalW. Meanwhile, The immunized mice were used to prepare their specific polyclonal antibodies. The immune cross-reactivity of the allergens was analyzed by Western blot analysis of the antibody against eight other crude protein extracts of crustacean food. Results: The identification results of mass spectrometry showed that the 21 000 allergen of Litopenaeus vannamei was a calcium-binding protein in muscle. The amino acid sequence homology analysis indicated that the allergen was consistent with that of Penaeus monodon, Penaeus chinensis, Procambarus clarkii, The sequence identity of SCP was 81% -100%. Western blot showed that the specific polyclonal antibodies against SCP were similar to those of Penaeus vannamei, Penaeus monodon, Penaeus monodon, Macrobrachium rosenbergii, Crawfish, sea crab, rust sturgeon, Chinese mitten crab extract in the SCP corresponding to about 21,000 reaction bands. Conclusion: The minor allergen of Mr 21 000 of Litopenaeus vannamei is a sarcoplasmic calcium-binding protein. The amino acid sequence has high homology with many crustaceans and a strong immune cross-reaction.