Production of recombinant insulin-like growth factor-II in the development of a radioimmunoassay in rainbow trout (Oncorhynchus mykiss).
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.
Gentil
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V
Laboratoire de Physiologie des Poissons, INRA, Rennes, France.
Martin
P
P
Smal
J
J
Le Bail
P Y
PY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000737:chemistry; D007335:Insulin-Like Growth Factor II / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011863:Radioimmunoassay / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II