| 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。
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| 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) |
| DOI：10.11693/hyhz201001013013 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目, 30670266 号 |
附件 |
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| PURIFICATION AND CHARACTERIZATION IDENTIFICATION OF VITELLOGENIN FROM PELTEOBAGRUS VACHELLI |
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LI Yu-Pei1,2, DIAO Xiao-Ming1, SHENG Xiao-Sa1, QUAN Heng1, ZHAI Xu-Liang1, LI Yun1
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1.Department of Fisheries Science, College of Animal Science and Technology, Southwest University, Key Laboratory of Reproductive and Developmental of Freshwater fish, Ministry of Education;2.Jiangsu Animal Husbandry & Veterinary College
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| Abstract: |
| 7-day after intraperitoneal injection of 17β-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 competitive 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.
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| Key words: Pelteobagrus vachelli, Vitellogenin, Purification, Polyclonal antiserum, Enzyme-linked immunosorbent assay (ELISA) |
