Cloning and expression of grass carp (Ctenopharyngodon idella) growth hormone
A cDNA fragment encoding grass carp (Ctenopharyngodon idella) growth hormone (gcGH) was identified by reverse transcription PCR, and the recombinant GH produced in E. coli JM 109 cells. Sequencing analysis confirmed that the nucleotide sequence of the PCR fragment was similar to a previously reported gcGH except two nucleotides which were mismatched with the coding region of the gcGH genomic gene (Zhu et al., 1992). This resulted in two changes in the deduced amino acid sequence: codon 107, GAG, coding for glutamine acid was changed to GTG; coding for valine, codon 138, CTG, coding for leucine was changed to CCG, coding for proline. Using recombinant gcGH, a polyclonal antibody was generated in rabbit for application in the study of GH production. Western blot analysis revealed that the polyclonal gcGH antibody specifically immunoreacted with recombinant gcGH, and with native GH from the pituitary extracts of grass carp, common carp, goldfish and zebrafish but not salmon, trout or tilapia, indicating general application of the antibody for GH studies in cyprinids. Immuno microscopic studies demonstrated that the gcGH antibody specifically reacted with somatotrophs in the pituitaries of grass carp and goldfish. Intraperitoneal injection of recombinant gcGH enhanced the growth rate of juvenile common carp demonstrating its biological activity. In vivo studies on GH production indicated that oestradiol administration increased pituitary GH levels in female goldfish. This was not associated with changes in steady-state pituitary GM mRNA levels, suggesting that oestradiol may enhance GH synthesis by post-transcriptional or post-translational mechanism. In vitro studies on GH gene regulation demonstrated that introns did not affect the gcGH expression efficiency in cultured fish non-pituitary cells.