Immunobiochemical study on growth hormone and vitellogenin in salmonids

从大麻哈鱼脑垂体中分离纯化生长激素。采用盐析、离子交换层析和凝胶过滤等方法对GH进行纯化。用重组鲑鱼生长激素（a-rsGH）免疫家兔获得抗血清，经SDS-PAGE和Western blotting鉴定，纯化蛋白为鲑鱼生长激素，并建立了特异、灵敏的鲑鱼生长激素低水平检测的酶联免疫吸附试验（ELISA）。以多克隆α-GH兔IgG为第一抗体，其生物素化的Fab'片段为第二抗体，通过抗生物素蛋白-生物素反应进行信号放大，通过夹心法进行非竞争性ELISA。该测定可在3天内运行，并在低至0.5 ng/ml的浓度下常规检测GH。GH的ELISA的发展使得定量血清GH水平成为可能。该ELISA与四种鲑属（虹鳟属、萨尔莫属、红点鲑属和哲罗鲑属）的脑垂体提取物和血清发生交叉反应。此外，纯化的鲑鱼生长激素和血清从几个物种的属Oncorhynchus的稀释曲线是平行的标准curve.During 5月，大约4个月前产卵，血清生长激素水平增加，达到峰值水平的马苏鲑鱼（O.masou）。此时体重也增加了。到6月，GH水平下降，并相对稳定，直到9月。从6月到9月，鱼的体重也保持不变。血清卵黄原蛋白（Vg）水平在5月上升，8月达到高峰水平，然后在9月下降。有趣的是，血清Vg水平的增加后，血清GH的增加发生在5月。

Growth hormone (GH) was purified from the pituitary glands of chum salmon (Oncorhynchus keta). The purification of GH was performed by saltingout, ion exchange chromatography and gel filtration. The purified protein was identified as chum salmon GH by SDS-PAGE and Western blotting using the antiserum obtained from a rabbit immunized with recombinant chum salmon GH (a-rsGH).A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum GH in chum salmon. The non-competitive ELISA was performed by a sandwich method using polyclonal a-GH rabbit IgG as the first antibody, its biotinylated Fab' fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made it possible to quantify of serum GH levels. This ELISA cross-reacted with pituitary extracts and sera from four salmonid genera (Oncorhynchus, Salmo, Salvelinus and Hucho). Moreover, dilution curves of purified chum salmon GH and sera from several species of the genus Oncorhynchus were parallel to the standard curve.During May, approximately four months prior to spawning, serum GH levels increased, reaching peak levels in Masu salmon (O.masou). At this time body weight also increased. By June, levels of GH declined and relatively stable until September. From June to September, body weight in the fish also remained constant. Serum vitellogenin (Vg) levels increased during May, reaching peak levels in August and then declined during September. Interestingly, the increase in serum Vg levels followed the increase in serum GH which occurred during May.