哲罗鲑生殖周期中血清卵黄蛋白原浓度的ELISA检测

Enzyme-linked immunosorbent assay(ELISA) of vitellogenin during reproductive cycle of taimen(Hucho taimen)

采用柱层析、蛋白免疫印迹和电泳等方法对哲罗鲑（Hucho taimen）的卵黄脂磷蛋白进行研究，并以其鼠抗和兔抗血清为抗体建立了夹心酶联免疫反应（ELISA）对生殖周期中哲罗鲑血清卵黄蛋白原浓度的变化进行检测。结果表明，哲罗鲑卵黄脂磷蛋白分子量在460ku，并且由3个亚基组成，分子量分别为137．8ku、84．2ku和10．8ku。蛋白免疫印迹表明，卵黄脂磷蛋白抗血清与雄鱼血清无特异性反应，而与雌鱼血清有特异的反应。本研究建立的双抗体夹心ELISA检测灵敏度为7．8ng／mL，批内变异系数为4．06％，批间变异系数为4．58％，工作范围为30～2000ng／mL，在该范围内标准曲线具有良好的线性和重复性。生殖周期中哲罗鲑血清卵黄蛋白原浓度由每年6月份产孵后的最低值573．4ng／mL升高到12月份的最高值1918．2ng／mL，卵黄积累持续期为6个月，整个生殖周期中血清卵黄蛋白原浓度只出现一个高峰期，表明人工养殖条件下，性成熟哲罗鲑的生殖周期为1年。

Lipovitellin was isolated from taimen （Hucho taimen ）eggs and Sandwich enzyme-linked immunosorbent assay （ELISA） was developed for quantification of vitellogenin （VTG）. The molecular weight was determined by Native-PAGE and gel filtration, and was 460 ku . With SDS-PAGE under reducing conditions, Lv was dissociated into 3 major subunits （137, 84 and 11 ku respectively）. Polyclonal antisera, produced against Lv , were reactive with all subunits in Western blots. The antisera were found to be highly cross-reactivity with female serum, no cross-reactivity with male by Western blotting. The detective coefficient of sandwich ELISA was 4.58% ,working range was 30 -2 000 ng/mL. In the range, the standard curve had good linearity and repeatability. In the reproductive cycle of taimen, serum vitellogenin level has increased from the 573 ng/mL on June to the highest 1 918 ng/mL on December each year, only one peak period, its showed that the reproductive cycle of Hucho taimen is one year.