Molecular characterization of goose-type lysozyme homologue of large yellow croaker and its involvement in immune response induced by trivalent bacterial vaccine as an acute-phase protein

Abstract

Lysozyme acts as an innate immunity molecule against the invasion of bacterial pathogens. Here, the cDNA of a goose-type lysozyme (g-lysozyme) was cloned from large yellow croaker ( Pseudosciana crocea) by expressed sequence tags (EST) and RACE-PCR techniques. The full-length cDNA of large yellow croaker g-lysozyme (LycGL) is 716 nucleotides (nt) encoding a protein of 193 amino acids (aa), with a theoretical molecular weight of 21.3 kDa. The deduced LycGL possessed the typical structural features of g-lysozyme, including three catalytic residues (E71, D84, D101) and four substrate binding sites (L97, L121, L128, G152). Genomic analysis revealed that the LycGL gene consisting of 2383 nt, contained five exons interrupted by four introns and exhibited a similar exon-intron organization to its homologues in Japanese flounder and Chinese perch, except for having a much longer intron 1 in the LycGL gene. Recombinant LycGL produced in Pichia pastoris exhibited obvious lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria such as Aeromonas sobria, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnficus. Tissue expression profile analysis showed that LycGL mRNA was constitutively expressed in all tissues examined, such as spleen, head kidney, intestine, liver, gills and heart, although at a different level. Upon stimulation with trivalent bacterial vaccine, LycGL mRNA levels in intestine, spleen and head kidney were quickly up-regulated and had 10.32-, 10.2- and 8.26-fold increases, respectively, and LycGL transcripts in intestine and head kidney reached their peak levels at 24 h post-induction and then decreased gradually while LycGL mRNA in spleen increased to its highest level at 48 h. These results suggest that LycGL may be involved in antibacterial immune response activated by bacterial vaccine as an acute-phase molecule.