瓦氏黄颡鱼(Pelteobagrus vachelli)卵黄蛋白原的纯化、性质鉴定及ELISA检测方法的建立  

PURIFICATION AND CHARACTERIZATION IDENTIFICATION OF VITELLOGENIN FROM PELTEOBAGRUS VACHELLI

作　　者：李育培[1,2] 刁晓明[1] 盛晓洒[1] 权恒[1] 翟旭亮[1] 李云[1] 

机构地区：[1]西南大学动物科技学院水产科学系淡水鱼类生殖与发育教育部重点实验室,重庆400716 [2]江苏畜牧兽医职业技术学院,泰州225300

出　　处：《海洋与湖沼》2010年第1期91-98,共8页Oceanologia Et Limnologia Sinica

基　　金：国家自然科学基金资助项目,30670266号

摘　　要：腹腔注射17β-雌二醇(E2),使瓦氏黄颡鱼雄鱼在7天内产生卵黄蛋白原(Vtg)。采用凝胶过滤和离子交换两种层析技术,从E2诱导的雄性瓦氏黄颡鱼血浆中分离、纯化出Vtg,采用糖、磷、脂蛋白染色技术证明分离、纯化的蛋白为Vtg,该Vtg在非变性条件下分子量约为240kDa,在SDS变性条件下分子量约为143kDa。纯化的瓦氏黄颡鱼Vtg经检测显示可能含有类胡萝卜素,但没有二硫键,对热相对稳定。利用纯化的瓦氏黄颡鱼Vtg,制备了兔抗瓦氏黄颡鱼Vtg多克隆抗血清。用双向免疫扩散法测得抗血清的纯度较高,效价为1︰32;Western blotting检测显示抗血清的特异性较好。以瓦氏黄颡鱼Vtg多克隆抗血清为抗体,以纯化的瓦氏黄颡鱼Vtg为抗原,建立了间接酶联免疫吸附反应(ELISA)方法检测瓦氏黄颡鱼体内Vtg的含量,标准曲线线性部分的线性方程为y=0.099x+0.4529(R2=0.9327),该方法检测的灵敏度为15.6ng/ml,工作范围为31.2—4000ng/ml,在此范围内,标准曲线具有良好的线性。7-day after intraperitoneal injection of 17 β -estradiol （E2）, male Pelteobagrus vachelli produced vitellogenin （Vtg）; and later the Vtg from the E2 treated P. vachelli plasma was isolated and purified by gel filtration and ion-exchange chromatography. With phosphor-, lipo- and glycol-protein staining methods, we verified this protein as Vtg, in molecular weight of about 240kDa detected by Native-PAGE. In SDS-PAGE, the Vtg broke into 2 same subunits, each at 143kDa. The purified Vtg contained carotenoid of non-disulfide bond, relatively stable to heat. To make use of purified Vtg, we prepared polyclonal antiserum against P. vachelli Vtg. Double immunodiffusion determined that the titre for Vtg antisera was 1 ： 32; and western-blotting demonstrated that polyclonal antiserum had preferably specific effect. An indirect com- petitive enzyme-linked immunosorbent assay （ELISA） has been then established for detecting the Vtg. The technique was developed using Vtg-resistant antiserum as antibody and Vtg as antigen in working range of 31.2--4000ng/ml, and the sensitivity at 15.6ng/ml. The equation linear part of a typical ELISA calibration curve is y =0.099x ＋ 0.4529 （R^2 = 0.9327）, which shows good linearity in the working range.

关 键 词：瓦氏黄颡鱼 卵黄蛋白原 纯化 多克隆抗血清 酶联免疫吸附反应(ELISA) 

分 类 号：S852.5[农业科学—基础兽医学] S965.325[农业科学—兽医学]