PROKARYOTIC EXPRESSION AND FUNCTION ANALYSIS OF LATEOLABRAX JAPONICA HEPCIDIN

The cysteine-rich peptide Hepcidin was known to be an antimicrobial peptide and iron transport regulator that had been found in mammals. Numerous teleost hepcidin sequences had been published and showed that Hepcidin was widespread among fish. The teleost Hepcidins probably had a function in the defense against invading bacteria as several reports demonstrate an upregulation of the gene expression after treatment with LPS, bacterins or live bacteria. A direct antibacterial effect of some of the synthetic teleost Hepcidins was also demonstrated. Farming of Lateolabrax japonica was a growing industry in china. However, bacterial and viral infections were contributing to reduced production. The goal of the present study was to molecularly clone the Lateolabrax japonica hepcidin (LjFishep) open reading frame (ORF), to express the LjFishep ORF in prokaryotic expression, to purify recombinant Lateolabrax japonica hepcidin (rLjFishep), and to detect the bioactivity of the rLjFishep in vitro. cDNA encoding LjFishep ORF was cloned by RT-PCR. Sequencing results showed that the LjFishep ORF nucleotide sequence was 261 bp in length, encoding a prepropeptide of 86 amino acids (aa) with a signal peptide of 24 aa. The predicted molecular weight of the peptide is 9.4 kD. A tentative RX(K/R)R motif for propeptide convertases was also identified suggesting a cleavage site located between Arg64 and Gla65. The deduced mature amino acid sequence of LjFishep was compared with those of the several avian and mammalian species. The results showed that the deduced mature rLjFishep amino acid sequence had 65%, 45.5% and 54.5% identities with Homo sapiens, Xenopus tropicalis and Danio rerio hepcidin in deduced mature amino acid sequence, and also had 82%?85% identities with other fish Hepcidin in deduced mature amino acid sequence. Phylogenetic analysis showed that the LjFishep had close relationship with Oplegnathus fasciatus and Perca fluviatilis hepcidin. Recombinant expression plasmid of pET-28a(+) was constructed by inserting the LjFishep ORF without the signal peptide sequence into the prokaryotic expression vector pET-28a(+). An expected protein band was observed on SDS-PAGE gel, recognized by monoclonal antibody against 6×His in western-blotting assay. In the pET-28a(+) expression system, many of the rLjFishep was found in inclusion bodies with a portion of soluble protein by SDS-PAGE. The monomer and multimers of soluble rLjFishep proteins were observed in native electrophoresis. The rLjFishep soluble protein was isolated by a quick protein isolation and purification system of ?KTA FPLC and identified by native PAGE. Bioactivity analysis showed that the rLjFishep monomer had the antimicrobial activity against Vibrio harveyi from Lateolabrax japonicain in a dose-dependent manner, and the minimal dose for inhibiting Vibrio harveyi was 6.25 μg/mL, it could establish a basis for further study the biological function and clinical application of Lateolabrax japonica Hepcidin.