Molecular identification and expression analysis of the CC chemokine gene in rock bream (Oplegnathus fasciatus) and the biological activity of the recombinant protein

Abstract

We identified the CC chemokine cDNA designated as RbCC1 (CC chemokine 1 in rock bream, Oplegnathus fasciatus), which was isolated using expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCC1 cDNA (850 bp) contained an open reading frame (ORF) of 366 bp encoding 122 amino acids. Results from our phylogenetic analysis demonstrated that the RbCC1 was closest relationship to the orange-spotted grouper and Mi-iyu croaker CC chemokines located within the fish CC chemokine group. RbCC1 was significantly expressed in the intestine, spleen, liver, and PBLs (peripheral blood leukocytes). Rock bream PBLs were stimulated with several mitogens, LPS and Con A/PMA which significantly induced the expression of RbCC1 mRNA in the PBLs. The RbCC1 mRNA expression in several tissues under conditions of bacterial and viral challenge was examined. The experimental challenge revealed that the kidney and spleen of fish infected with Streptococcus iniae showed the most significant increases in RbCC1 expression compared to the control. In the case of RSIV infection, the RbCC1 mRNA expression was markedly up-regulated in the liver. In this study, recombinant RbCC1 (approximately 53 kDa) was produced using an Escherichia coli expression system followed by purification. Subsequently, the addition of purified rRbCC1 was examined to investigate the impact on the proliferative and chemotactic activity on kidney leukocytes from rock bream. The results demonstrated that the rRbCC1 induces significant biological activity on kidney leukocyte proliferation and attraction at concentrations in the range of 10-300 μg/mL and suggests that rRbCC1 could be utilized as an immune-stimulant and/or molecular adjuvant to enhance the immune effects of vaccines.