Cloning, expression and antimicrobial activity of Hepcidin from Hypophthalmichthys molitrix

The full-length Hepcidin cDNA sequence GenBank No.KF312213 was amplified from the liver of chub (Hypophthalmichthys molitrix) by semi-Nested PCR rapid amplification of cDNA ends (RACE).According to prodomain and mature peptide of the Hepcidin cDNA sequence, we designed an upstream primer with EcoR I restriction site and downstream primers with Sal I restriction site, and cloned the target gene into the expression vector pET-32a (+). The recombined plasmid was transformed into the expression stain-E .coliRosetta and expressed induciblely at different temperatures (37 ℃, 28 ℃ and 16 ℃) and of different IPTG concentrations (0.5 mmol · L^-1 and 1.0 mmol · L^-1). The protein was purified by Ni Sepharose^TM affinity chromatography column. The full-length of the Hepcidin cDNA gene was 755 bp, which included a 282-bp ORF encoding a 93-amino acid prepropeptide. The prepropeptide contained a signal peptide (24 amino acids), a prodomain (42 amino acids) and a mature peptide (27 amino acids). The purified product displayed a single protein band through 15% SDS-PAGE electrophoresis. The purified production of pET-Hep/Rosetta had obvious antibacterial effect on Streptococcus agalactiae, Staphylococcus aureus and Aeromonas hydrophila, but the control group showed no inhibitory effect.