抄録
Vitellogenin (Vtg), the egg yolk precursor protein, was purified from plasma of estradiol-17β (E2) -treated juvenile Koi carp to homogeneity. The native Koi carp Vtg with a molecular mass of 300 kDa was a homodimer of an identical subunit with a molecular mass of 150 kDa. Absorbance at 280 nm and phosphorus content of the Vtg were 0.890/cm per mg of protein and 0.234µmol/mg of protein, respectively and are similar to those reported on gold fish Vtg. The N-terminus of the Koi carp Vtg was blocked, and deblocking was accomplished by digestion with pyroglutarnate aminopeptidase, suggesting that the N-terminal residue was pyroglutamate. The sequence of 30 amino acids after removal of the pyroglutamate was 59-83% identical to those of Vtgs from various fishes. The sequences of 2 internal peptides obtained by a digestion with V8 protease were 64-90% identical to those in internal regions of Vtgs from fathead minnow or rainbow trout. Ouchterlony and Western blot analysis demonstrated high specificity of a polyclonal antiserum raised in rabbit against the purified Koi carp Vtg. A homologous enzyme-linked immunosorbent assay (ELISA) was established using the antiserum and the Koi carp Vtg. The lowest detectable concentration by the ELISA was 10 ng/ml (at 95% binding), The intra- and inter-assay variations were respectively 4.5% and 7.2% at around 60% binding and those were 8.8 % and 14.6 % at around 95% binding. The ELISA was well applicable to determination of minute concentrations of Vtg in juvenile and male Koi carp plasma. The plasma Vtg levels in the juvenile Kai carp and the E2-treated juvenile Koi carp were 0.41 ± 0.12µg/ml (n = 30) and 57.2 ± 10.4 mg/ml (n = 11), respectively.
