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

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

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。

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.