鲢骨骼肌过敏原小清蛋白的分离纯化及多克隆抗体制备与应用

Purification and polyclonal antibody preparation of parvalbumin from silver carp(Hypophthalmichthy molitrix) skeletal muscle

小清蛋白是鱼类的主要过敏原,对该蛋白的研究不仅有利于过敏原检测方法的建立也可为低致敏性水产品的开发提供理论依据。通过组织捣碎、冷冻离心、热处理、Superdex75凝胶过滤等方法从鲢白色肉中纯化得到过敏原小清蛋白。Tricine-SDS-PAGE显示,在非还原条件下,该蛋白呈分子量分别为12ku、14ku、24ku的3个条带。而在还原条件下,仅有分子量为12ku的条带。Western-blotting分析表明,分子量为12ku、14ku和24ku的这3个条带都与小鼠抗蛙小清蛋白单克隆抗体(PARV-19)发生特异性反应,提示它们均为小清蛋白的不同形态。用纯化的小清蛋白制备多克隆抗体,经Protein A Sepharose亲和层析纯化得到高纯度的免疫球蛋白G(IgG)。Dot-blot检测发现,抗体稀释至1/51200时仍能与纯化的小清蛋白有显色反应。用制备的多克隆抗体进行Western-blotting分析,能特异地检测4种鱼(鲤、鲢、鲫、黄鳍鲷)中的小清蛋白。

Parvalbumin is the major allergen of fish species. Investigation into this protein is beneficial not only to detecting allergen, but also to producing aquatic products with low allergenicity. In the present study, parvalbumin from the skeletal muscle of silver carp （Hypophthalmichthy molitrix） was first purified to homogeneity by homogenization, centrifugation, heat treatment extraction and gel filtration chromatography on Superdex 75. Purified parvalbumin revealed three protein bands with molecular mass of 12, 14 and 24 ku, respectively, as detected by Tricine-SDS-PAGE under non-reducing conditions while under reducing conditions, only a band corresponding to 12 ku was detected. Western-blotting using anti-frog parvalbumin monoclonal antibody （PARV-19） positively reacted with the three protein bands of 12, 14 and 24 ku, suggesting they are different forms of parvalbumin. A polyclonal rabbit anti-silver carp parvalbumin antibody was prepared and immunoglobulin G （IgG） was purified by Protein A Sepharose affinity column. Dot-blot revealed that the antibody reacted with parvalbumin even after dilution to 1/51200, suggesting its higher titer. Western-blotting analysis indicated that parvalbumins from four fishes （common carp, silver carp, crucian carp, sea bream） can all be detected by the polyclonal antibody specifically.