Construction of cDNA library of gills from Pagrus major with the cloning of hepcidin

With the layers of mucus and nonspecific immune defenses, the fish gill is exposed to the external environment and forms the initial barrier to invasio n by pathogens. As one of the mucosa-associated lymphoid tissues in teleost fishes, gill contains populations of leucocytes. In order to probe the immune-related genes in red seabream (Pagrus major), the cDNA library of gill s was constructed from the red seabream inoculated with several species of pathogenic bacteria. The total RNA was extracted by TRIZOL and mRNA was isolated by biotin-oligo (dT). Then, cDNA was synthesized from 5 μg mRNA. Through running gel electrophoresis of double-strand cDNA, the fragments of 500-4000 bp were harvested, concentrated, ligated with λZAP vector and packaged in vitro. The primary cDNA library contained 1.75 ×10⁵ recombinants. The titer of amplified library was tested up to 1×10⁹ pfu·mL^-1, and the insert size of random picked phagemid was between 500-2000 bp. PCR with specific primers of hepcidin was performed to cloning of the homologous genes from cDNA library and a fragment of hepcidin cDNA sequence was amplified. The identity was above 85% in comparison with the hepcidin cDNA isolated from the liver of also red seabream. Therefore, the construction of cDNA library provides the basis for screening immune-related genes from red seabream.