食品科学 ›› 2019, Vol. 40 ›› Issue (12): 308-314.doi: 10.7506/spkx1002-6630-20180706-084

• 安全检测 • 上一篇    下一篇

牙鲆(Paralichthys olivaceus)中不同亚型小清蛋白的提纯与鉴定

孙礼瑞1,林 洪1,李振兴1,*,赵金霞2,林 航2,罗 晨1,田盛兰1   

  1. 1.中国海洋大学食品科学与工程学院,山东 青岛 266003;2.青岛大学附属医院,山东 青岛 266003
  • 出版日期:2019-06-25 发布日期:2019-06-28
  • 基金资助:
    中央高校基本科研业务费专项(201762004)

Purification and Characterization of Parvalbumin Isotypes from Japanese Flounder (Paralichthys olivaceus)

SUN Lirui1, LIN Hong1, LI Zhenxing1,*, ZHAO Jinxia2, LIN Hang2, LUO Chen1, TIAN Shenglan1   

  1. 1. College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China; 2. The Affiliated Hospital of Qingdao University, Qingdao 266003, China
  • Online:2019-06-25 Published:2019-06-28

摘要: 以牙鲆(Paralichthys olivaceus)为研究对象,以Tris、甘氨酸和二硫苏糖醇的混合溶液为提取液,加入不同量的硫酸铵分级纯化小清蛋白,应用免疫印迹法、酶联免疫法及质谱法进行鉴定。最终提纯得到3 种不同分子质量的蛋白(PVI、PVII和PVIII),所得蛋白纯度大于90%,血清学实验证明其与小清蛋白特异性IgG及IgE抗体均有结合能力,质谱鉴定得到PVII和PVIII分子质量分别为12 151 Da和11 645 Da,pI值分别为5.14和4.69。该方法满足牙鲆小清蛋白提取纯化要求,得到的3 种蛋白是牙鲆小清蛋白的不同亚型,且均具有过敏原性。

关键词: 鱼过敏原, 小清蛋白亚型, 牙鲆, 纯化, 鉴定

Abstract: Parvalbumins were extracted and purified from the muscle of Japanese flounder (Paralichthys olivaceus). A mixed solution of Tris, glycine, and DL-dithiothreitol was used as the extraction solvent. Parvalbumins were purified by adding ammonium sulfate to different degrees of saturation, and then identified using enzyme-linked immunosorbent assay (ELISA), Western-blot and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Three proteins (PVI, PVII and PVIII) with different molecular masses were obtained with high purity ( > 90%). Serological results demonstrated that each of these parvalbumins had the ability of binding to the specific IgG and IgE. The molecular masses of PVII and PVIII were determined to be 12 151 and 11 645 Da with Isoelectric point (pI) of 5.14 and 4.69, respectively. This method can meet the requirements for the extraction and purification of parvalbumins. These proteins were proved to represent different isotypes of parvalbumins with allergenicity.

Key words: fish allergen, parvalbumin isotypes, Japanese flounder, purification, identification

中图分类号: