Gentil V, Martin P, Smal J, Le Bail P Y
Laboratoire de Physiologie des Poissons, INRA, Rennes, France.
Gen Comp Endocrinol. 1996 Nov;104(2):156-67. doi: 10.1006/gcen.1996.0158.
Rainbow trout (Oncorhynchus mykiss) insulin-like growth factor-II (IGF-II) cDNA was amplified by reverse transcription-polymerase chain reaction and cloned into an expression vector (parHS3) for production by Escherichia coli. Recombinant rainbow trout IGF-II (rtIGF-II) was recovered from bacterial inclusion bodies, solubilized, and purified by gel filtration chromatography under reducing conditions. After refolding of the peptide by dialysis at basic pH, we obtained a final yield of 820 micrograms/liter culture medium. The rtIGF-II appeared as a single band estimated at 80% and with molecular mass of 7.5 kDa. A homologous radioimmunoassay (RIA) for the measurement of IGF-II in trout plasma has been developed using rtIGF-II as antigen, radiolabeled tracer, and standard. RIA sensitivity was 0.1 ng/ml and the ED50 was 0.75 +/- 0.06 ng/ml. Intra- and interassay coefficients of variation were 4.04% (n = 6) and 4.56% (n = 9), respectively, at ED50 levels. Cross-reactivities of 1 and 0.1% were found for recombinant coho salmon (Oncorhynchus kisutch) IGF-I and coho salmon insulin, respectively. No cross-reactivities were found for recombinant human IGF-I and IGF-II, porcine insulin, or a variety of salmonid pituitary hormones. The interference of IGF binding proteins (IGFBP) in the RIA has been tested with and without extraction (acid/ethanol extraction or C-25 acid chromatography) of fed and fasted trout plasma samples. Parallel displacement curves were obtained for unextracted and C-25-extracted plasma but not for acid/ethanol-extracted plasma. Comparison of IGF-II levels from these samples showed a possible interference of IGFBP in the RIA in fed (19.6 +/- 1.56 ng/ml unextracted and 25.25 +/- 1.21 ng/ml extracted) and fasted (21.58 +/- 1.02 ng/ml unextracted and 9.86 +/- 1.68 ng/ml extracted) trout plasma. Finally, the displacement curves for unextracted plasma from rainbow trout, Couch's sea bream (Pagrus pagrus), and carp (Cyprinus carpio) were parallel to that of the rtIGF-II standard and not strictly parallel for tilapia (Oreochromis niloticus) and catfish (Silurus glanis) plasma. No significant cross-reaction occurred with eel (Anguilla anguilla) plasma. The rainbow trout IGF-II RIA reported in this study has low variability and is highly sensitive. Therefore it is a reliable method for measuring IGF-II levels in salmonids and some nonsalmonid fish species.
通过逆转录-聚合酶链反应扩增虹鳟（Oncorhynchus mykiss）胰岛素样生长因子-II（IGF-II）cDNA，并将其克隆到表达载体（parHS3）中，以便由大肠杆菌生产。从细菌包涵体中回收重组虹鳟IGF-II（rtIGF-II），在还原条件下通过凝胶过滤色谱法使其溶解并纯化。在碱性pH下通过透析对该肽进行复性后，我们获得了820微克/升培养基的最终产量。rtIGF-II呈现为一条单一的条带，估计纯度为80%，分子量为7.5 kDa。已开发出一种同源放射免疫测定法（RIA），以rtIGF-II作为抗原、放射性标记示踪剂和标准品来测量鳟鱼血浆中的IGF-II。RIA的灵敏度为0.1纳克/毫升，半数有效剂量（ED50）为0.75±0.06纳克/毫升。在ED50水平下，批内和批间变异系数分别为4.04%（n = 6）和4.56%（n = 9）。重组银大麻哈鱼（Oncorhynchus kisutch）IGF-I和银大麻哈鱼胰岛素的交叉反应率分别为1%和0.1%。未发现重组人IGF-I和IGF-II、猪胰岛素或多种鲑鱼垂体激素有交叉反应。已在喂食和禁食的鳟鱼血浆样品进行或未进行提取（酸/乙醇提取或C-25酸色谱法）的情况下，测试了IGF结合蛋白（IGFBP）对RIA的干扰。未提取和C-25提取的血浆获得了平行的置换曲线，但酸/乙醇提取的血浆未获得。这些样品中IGF-II水平的比较表明，IGFBP可能对喂食（未提取的为19.6±1.56纳克/毫升，提取的为25.25±1.21纳克/毫升）和禁食（未提取的为21.58±1.02纳克/毫升，提取的为9.86±1.68纳克/毫升）的鳟鱼血浆中的RIA产生干扰。最后，虹鳟、黑鲷（Pagrus pagrus）和鲤鱼（Cyprinus carpio）未提取血浆的置换曲线与rtIGF-II标准品的平行，而罗非鱼（Oreochromis niloticus）和鲶鱼（Silurus glanis）血浆的置换曲线并非严格平行。鳗鱼（Anguilla anguilla）血浆未发生明显交叉反应。本研究中报道的虹鳟IGF-II RIA变异度低且灵敏度高。因此，它是测量鲑鱼和一些非鲑鱼鱼类中IGF-II水平的可靠方法。
