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

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。

PURIFICATION AND CHARACTERIZATION IDENTIFICATION OF VITELLOGENIN FROM PELTEOBAGRUS VACHELLI

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(R2=0.9327),which shows good linearity in the working range.