Funkenstein B, Cohen I
Israel Oceanographic & Limnological Research Institute, Tel Shikmona, Haifa, Israel.
Growth Regul. 1996 Mar;6(1):16-21.
The ontogeny of growth hormone (GH) protein and mRNA was studied in the gilthead sea bream Sparus aurata by Northern blot, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Total RNA was prepared from a pool of larvae collected on different days after hatching. When hybridized to Sparus aurata GH cDNA, GH specific mRNA was first seen on day 3 post-hatching. Levels increased on subsequent days and reached the highest levels on day 9; thereafter a decrease was noted to similar levels between days 10 and 16. RT-PCR amplification of larval RNA showed an amplified fragment on day 3, which hybridized to Sparus aurata GH cDNA but not on days 1 and 2 after hatching. Soluble proteins were electrophoresed and immunoreacted with antibodies raised against purified Sparus GH. A faint band was detected on day 2 after hatching, which had an electrophoretic mobility very similar to recombinant tilapia GH or Sparus pituitary extract. The immunoreactive material increased on days 3, 4 and 6. Specificity of the immunoreaction was demonstrated by the ability of recombinant tilapia GH to complete with GH bound to the membrane and inability of normal rabbit serum (NRS) to detect GH on the membrane. Our results suggest that both GH mRNA and protein are expressed in Sparus aurata shortly after hatching.
通过Northern印迹、逆转录-聚合酶链反应（RT-PCR）和Western印迹分析，研究了金头鲷（Sparus aurata）生长激素（GH）蛋白和mRNA的个体发生。从孵化后不同天数收集的幼虫池中制备总RNA。当与金头鲷GH cDNA杂交时，在孵化后第3天首次观察到GH特异性mRNA。随后几天水平升高，在第9天达到最高水平；此后，在第10天至第16天之间观察到下降至相似水平。幼虫RNA的RT-PCR扩增在第3天显示出一个扩增片段，该片段与金头鲷GH cDNA杂交，但在孵化后第1天和第2天未出现。对可溶性蛋白进行电泳，并与针对纯化的金头鲷GH产生的抗体进行免疫反应。在孵化后第2天检测到一条 faint带，其电泳迁移率与重组罗非鱼GH或金头鲷垂体提取物非常相似。免疫反应性物质在第3、4和6天增加。重组罗非鱼GH与膜结合的GH竞争的能力以及正常兔血清（NRS）在膜上检测不到GH的能力证明了免疫反应的特异性。我们的结果表明，GH mRNA和蛋白在金头鲷孵化后不久就开始表达。
