Cloning, Expression And Antimicrobial Activity Analysis Of Hepcidin Gene Variants In Two Cultured Fish, Pagrus Major And Spagrus Macrocephalus

As a major part of immediately effective, nonspecific defenses against infections, antimicrobial peptides (AMPs) play a crucial role in the innate immune system. Hepcidin is a circulating antimicrobial peptide and putative iron regulatory hormone previously described in mice and humans. There are several species of fish hepcidins that have been isolated and characterized so far. It was speculated that hepcidin might spread wildly among fish. Fishes are in intimate contact with a rich microbial flora and exposed to millions of potential pathogens daily, through ingestion and inhalation, so they have a tendency to use their innate immune system as the first line of defense against microbial invasion. Thus, isolation and characterization of AMPs like hepcidin will facilitate to further elucidate the innate mechanism of fish to survive healthy in the complex marine environment. In this study, two species of commercial maricultured fish - red seabream (Pagrus major) and black porgy (Spagrus macrocephalus) were taken as samples to be used to study on hepcidin gene cloning, tissue-specific distribution, in vitro protein expression and antibacterial activities. Meanwhile, due to the high number of cysteines, synthesis of hepcidin might be difficult. Therefore, an in vitro Escherichia coli expression system was developed based on the previous results in order to obtain enough quantities of hepcidins to be used for analysis of the antimicrobial activities or other functions. The results are showed as follows:1 As one of the mucosa-associated lymphoid tissues in teleost fishes, gills form the initial barrier to invasion by pathogens. In order to probe and isolate the antimicrobial peptide and other immune-related genes, a cDNA library of gills was constructed from the red seabream infected with pathogenic bacteria. After total RNA was extracted and mRNA isolated, the cDNA was synthesized. Through running gel electrophoresis of double-strand cDNA, the fragments of 500bp~4000bp were harvested, concentrated, ligated withλZAP vector and packaged in vitro. The primary cDNA library contained～1.75×105 recombinants. The titer of amplified library was...