Cloning, Recombinant Expression And Antibacterial Activity Of A Hepcidin Gene From Epinephelus. Awora

A pair of PCR primer was designed according to the antimicrobial peptide Hepcidin gene of Lateolabrax sp. and a fragment of gene was cloned from the liver of Epinephelus. Awora. To obtained the full-length cDNA sequence of the gene, rapid amplification of cDNA ends (RACE) was used. The full-length cDNA of the gene is 592 nucleotides, which includes a 261 bp ORF encoding a 87-amino acid prepropeptide. the prepropeptide contains a signal peptide (24 amino acids), a prodomain (40 amino acids) and a mature peptide (23 amino acids, with 4 cysteine residues). The mean molecular weight and the theoretical pI of the mature peptide is 2465.8 Da and 5.97 respectively. BLAST in GenBank database reveals that the prepropeptide is different from any other reported antimicrobial peptides. But there is some similarity between the amino acid sequences encoded by the ORF and Hepcidin from some fish,and is predicted as a member of Hepcidin.Reverse PCR was used for cloning the genomic DNA sequence of the gene. The genomic DNA sequence of this gene is similar to other fish, which contains three exons and two introns. The first exon contains 5'UTR, signal peptide and partial prodomain sequences. The second exon only encodes some part of prodomain, and the third exon encodes 3 'UTR and partial prodomain. Furthermore, general upstream elements of the gene were cloned, which include TATA box and a capping signal. In addition, several consensus-binding motifs for transcription factors were also found in the 5' flanking sequences of this Hepcidin.The ORF sequence without signal peptide was recombined into pTrc-CKS vector and expressed in Escherichia coli. The expressed protein mainly existed in inclusion body. The fusion proteins were purified by Ni-NTA column. The pure fusion protein was digested with 3C protease.A 14 kDa objective protein was obtained. Antibacterial activity of the objective protein was tested. The results showed that the protein had obviously antibacterial activity against both Gram-positive and Gram-negative bacterial. Heated under 65℃for 60 min, the protein still has strong antibacterial activity against some bacteria, which indicated that it is a heat-stable protein.In conclusion, the gene structure and biological function of an antimicrobial peptide belonging to the family of Hepcidin have been characterized. This is the first report on characterization of a novel anionic antimicrobial peptide Hepcidin gene from the liver of E.awora. This study also lays foundation for the study of gene recombinant engeering production of antimicrobial peptide and the application of this antimicrobial peptide.