Immunological response of goldfish (Carassius auratus) to recombinant pro-hepcidin and its antibacterial activity against Aeromonas hydrophila

Hepcidin is a key antimicrobial peptide involved in innate immunity in fish. The present study aimed to produce recombinant pro-hepcidin from goldfish (Carassius auratus) (rpro-CuHep) and to evaluate its antibacterial activity as well as its effects on immune responses and resistance against Aeromonas hydrophila infection in goldfish. The full-length goldfish pro-hepcidin gene was synthesized using codon optimization for Escherichia coli BL21 Rosetta and cloned into the pET28a + expression vector. Successful transformation was confirmed by Colony PCR, yielding a 287-bp fragment. Recombinant protein expression was induced with 1 mM IPTG at 37 °C for 8 h. The rpro-CuHep peptide was purified using affinity chromatography and identified as an approximately 10-kDa protein by SDS-PAGE and Western blot analysis. Antibacterial assays demonstrated that rpro-CuHep exhibited notable inhibitory activity against A. hydrophila and Streptococcus iniae. In vivo challenge experiments showed that goldfish treated with rpro-CuHep had significantly lower mortality rates following A. hydrophila infection compared to the control group. Furthermore, immune parameters including total protein, lysozyme activity, alternative complement activity (ACH50), and total immunoglobulin levels were significantly elevated in rpro-CuHep–treated fish (p < 0.05). Gene expression analysis revealed significant upregulation of interleukin-6 (IL-6) and interleukin-10 (IL-10) (p < 0.05), whereas no significant change was observed in interleukin-8 (IL-8) expression (p > 0.05). The results demonstrate that rpro-CuHep possesses strong antibacterial activity and can significantly enhance immune responses and disease resistance in goldfish. These findings suggest that recombinant pro-hepcidin has potential as an effective immunostimulant and antibacterial agent for controlling bacterial infections such as A. hydrophila in aquaculture.