Molecular and functional characterization of a novel CC chemokine in large yellow croaker (Pseudosciaena crocea)

A full-length CC chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCC2). Its open reading frame (ORF) is 282 nucleotides (nt), encoding a polypeptide of 93 amino acids (aa). The deduced LycCC2 contains a 20-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CC chemokines (C(34), C(35), C(61) and C(75)). It shares 17.4-40.4% and 10.0-24.5% aa sequence identities to other known bony fish and mammalian CC chemokines, respectively. Tissue expression distribution analysis showed that LycCC2 gene was constitutively expressed in eight of all nine tissues examined, except for liver. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, LycCC2 gene expression was obviously up-regulated in blood, kidney, heart, spleen, intestine and gills at 12 h post-induction, and also induced in liver. The time-course analysis using a real-time PCR showed that LycCC2 transcript in spleen and kidney was quickly increased by either poly(I:C) or bacterial vaccine and reached peak levels at 12h, followed by a recovery to normal level after 48h. LycCC2 expression in spleen and kidney was more strongly up-regulated by bacterial vaccine (37.3- and 17.4-fold mRNA increases at 12h post-induction, respectively) than by poly(I:C) (15.5- and 8.5-fold mRNA increases, respectively). Recombinant LycCC2 (rLycCC2) protein produced in Pichia pastoris exhibited marked chemotactic ability for the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggested that LycCC2 represents a novel member of the fish CC chemokine family, which may be involved in a pro-inflammatory function in the immune response triggered by bacterial vaccine or poly(I:C) in large yellow croaker.