摘要
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.
出处
《上海海洋大学学报》
CAS
CSCD
北大核心
2009年第6期673-679,共7页
Journal of Shanghai Ocean University
基金
国家支撑计划(2006BAD03B08-01)