尼罗罗非鱼卵黄脂磷蛋白的分离纯化与性质鉴定  

Purification, identification, and characterization of lipovitellin from tilapia(Oreochromis niloticus)

作　　者：单瑞后 王松[2] 王骏[3] 王军[1] 汝少国[1] 

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]中国水产科学研究院黄海水产研究所,山东青岛266071 [3]山东出入境检验检疫局,山东青岛266002

出　　处：《中国水产科学》2015年第4期638-644,共7页Journal of Fishery Sciences of China

基　　金：国家质检总局课题计划项目(2013IK196)

摘　　要：采用Sephacryl S-300过滤层析和DEAE-Sepharose Fast Flow离子交换层析相结合的方法从尼罗罗非鱼(Oreochromis niloticus)成熟卵子匀浆液中分离纯化出了一种高分子量的蛋白。该蛋白能被Schiff试剂、甲基绿和苏丹黑B着色,Western blot显示能被金鱼卵黄脂磷蛋白(lipovitellin,Lv)多克隆抗血清特异性识别,在非变性条件下分子量约为560 k D,在SDS变性条件下分子量约为112 k D,结果表明分离纯化的蛋白是一种含有糖、磷、脂基团的蛋白,符合鱼类Lv的性质,且与金鱼Lv有免疫交叉反应,从蛋白的性质和免疫原性以及分子量大小等角度判断,本研究获得的高纯度蛋白为尼罗罗非鱼卵黄脂磷蛋白;纯化的罗非鱼Lv在反复冻融、37℃及60℃处理条件下均未出现降解,表明罗非鱼Lv比鱼类卵黄原蛋白(Vitellogenin,Vtg)更为稳定。研究结果为罗非鱼Lv抗体的制备奠定了基础。Lipovitellin （Lv） is the major proteolytic product of vitellogenin （Vtg） in the ovary and has been widely used in studies of endocrine disruption. In this paper, a high molecular weight protein was purified from the ovary extracts of Nile tilapia （Oreochromis niloticus） using gel filtration followed by ion-exchange chromatography. Results of native polyacrylamide gel electrophoresis （PAGE）, lipid staining with Sudan black B, carbohydratestaining with Schiff reagent, and phosphorus staining with methyl green identified the purified protein as a phosphoglycoprotein. Using western blotting, the protein was cross-reacted with the anti-goldfish Lv antisera. Native PAGE determined the molecular weight of the protein to be approximately 560 kD, and a single monomer of ~112 kD was detected by the sodium do-decyl sulfate （SDS）-PAGE. Based on this characterization, immunogenicity, and molecular weight, this protein was identified as the Lv of the Nile tilapia. No degradation was observed under conditions of multigelation or incubation at 37℃ or 60℃; thus, the purified Nile tilapia Lv exhibited a relatively higher stability than vitellogenin. To our know-ledge, this is the first report of the purification and characterization of the Nile tilapia Lv. Results of the present study provide a theoretical foundation for the manufacture of polyclonal antisera against Nile tilapia Lv which could then be used to detect vitellogenin induction in tilapia.

关 键 词：尼罗罗非鱼 卵黄脂磷蛋白 纯化 性质鉴定 

分 类 号：S94[农业科学—水产养殖]