Abstract

To make up the flaw that there is no available information about ghrelin gene in crucian carp. The ghrelin gene was amplified by reverse transcription-PCR (RT-PCR) using total RNA extracted from intestine of crucian carp. PCR product was cloned into the pMD®19-T vector to construct pMD®19-T-ghrlein for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a and was transformed into hostEscherichia coli strain Rosetta for expression. In this study, 490 bp fragment of ghrelinwas obtained by RT-PCR. In comparison with other fishes, the amino acid sequences ofghrelin in crucian carp showed a high similarity to that of goldfish (99%). The high expression of ghrelin gene was detected in the intestine and liver by real-time PCR. IPTG at concentrations of 0, 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 mmol/L could efficiently induce the expression of pGh-32. The result showed that the optimal concentration of IPTG was 0.3 mmol/L by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The ghrelin gene expressed as early as 1 h after IPTG induction, and reached peak levels after 3 h. Successful expression of ghrelin fusion protein in prokaryotic cell could lay a basis for further study of industrial production.

Key words: Crucian carp, ghrelin, cloning, prokaryotic expression.