Molecular cloning, characterization, and expression analysis of a disease-resistance related CC chemokine gene in miiuy croaker (Miichthys miiuy)
Introduction
The main role of the innate immune system is believed to be the first line of host defense in opposing pathogenic organisms and to deal with any foreign material until the adaptive immune system is able and potent enough to take over (Janeway and Travers, 1997, Holland and Lambris, 2002, Sinyakov et al., 2002). In fish, the innate immune response has been considered an essential component in combating disease incidents due to the constraints placed on the adaptive immune response by their poikilothermic nature plus the limited antibody repertoires, affinity maturation and memory and relatively slow lymphocyte proliferation (Magnadottir, 2006).
A cascade of pro-inflammatory cytokines including chemokines is released as important part of the non-specific innate immune response (Whyte, 2007). Chemokines are small, structurally related and secreted peptides that play a crucial role in stimulating the recruitment, activation, and adhesion of cells to sites of infection or injury (Neville et al., 1997, Laing and Secombes, 2004a). In general, chemokine ligands engage specific receptors on the leukocyte surface which contain seven membrane-spanning α helices and are coupled to GTP-binding protein (Murdoch and Finn, 2000). Chemokines are under particularly strong selection and are one of the eight most rapidly changing proteins and domains (Waterston et al., 2002). At present more than 50 mammalian chemokines have been reported and major of them contain four conserved cysteine residues that form intramolecular disulfide bonds. According to the arrangement of the first two cysteine residues localized proximally to the N-terminus of molecular, chemokines are divided into four groups: CXC, CC, C and CX3C. CC chemokines constitute the largest subfamily of chemokines in mammalian species with 28 members (Bacon et al., 2003). They share a tertiary structure containing three β-strands and one α helices at the C-terminal and are considered to act on mononuclear cells rather than on neutrophils for CXC chemokines (Laing and Secombes, 2004a).
Much interest has been generated in the study of fish chemokines in recent years. As a result, a number of sequences have been described for chemokines and their receptors in various fish species, such as one in carp (Fujiki et al., 1999), five in Japanese flounders (Khattiya et al., 2003, Khattiya et al., 2004, Khattiya et al., 2007, Kono et al., 2003), 7 CC chemokines in cichlid fish (Kuroda et al., 2003), 18 CC chemokines in rainbow trout (Dixon et al., 1998, Liu et al., 2002, Laing and Secombes, 2004b), and 26 CC chemokines in channel catfish and blue catfish (He et al., 2004, Peatman et al., 2005). In zebrafish, 46 loci were found to encode putative CC chemokines (Peatman and Liu, 2006). In salmon, 30 putative CC chemokines have been screened from 466 ESTs (Peatman and Liu, 2007).
Miiuy croaker, Miichthys miiuy, is a marine economical fish that is mainly distributed from the western Japan Sea to the East China Sea. In China, it is very important food fish specie with a worldwide market demand, due to its good taste and high nutritive and medicinal value, and has been widely cultured since the late 1990s (Lou, 2004). As fish culture is associated with diseases, protection of fish against pathogens is the major challenge faced by fish health scientists. The use of antibiotics has partially solved the problem, but has raised concerns regarding drug residue and environmental pollution. It is necessary to breeding new miiuy croaker stock with enhanced disease resistance by using marked-assisted selection. Thus, cloning and molecular characterization of the CC chemokine gene from miiuy croaker are very important for genetic selection of this fish species.
In the present study, we cloned and characterized a new CC chemokine cDNA and its gene from miiuy croaker, designated as MimiCC2, and we built homology model of protein of the MimiCC2 chemokine gene. We also examined the tissue distribution of MimiCC2 mRNA and analyzed modulation of MimiCC2 gene expression upon stimulation with menacing bacteria Vibrio anguillarum.
Section snippets
Fish sampling and challenge experiments
Healthy miiuy croakers (mean weight 800 g) were obtained from Zhoushan Fisheries Research Institute (Zhejiang, China). Ten tissues (liver, spleen, kidney, intestines, heart, muscle, stomach, brain, swim bladder, and fin) of three uninfected miiuy croakers were removed and kept separately at − 80 °C until use.
After one week of acclimatizing in aerated seawater tanks, these fish were used for the challenge experiments. Briefly, V. anguillarum were cultured at 28 °C to mid-logarithmic growth in marine
Cloning, characteristics and homology model of MimiCC2
The full length cDNA of MimiCC2 is 1016 bp, including a 5′-untranslated region (UTR) of 94 bp and a 3′-UTR of 622 bp. The 300 bp open reading frame of MimiCC2 encodes a protein of 99 amino acids residues. The N-terminal segment included a high proportion of hydrophobic amino acid residues, and therefore, the first 23 amino acid residues were predicted to be a signal peptide by the SignalP program, which indicates MimiCC2 chemokine is a secreted molecule and the mature protein contains 76 amino
Discussion
Innate immunity is widely believed to play a very important role in resistance against major bacterial disease in fish. As one of the innate immune responses, chemokines are key components in the process of leukocyte recruitment in inflammatory sites. The interactions of various chemokines with their receptors on leukocytes allow activation and chemotaxis of neutrophils, eosinophils, lympgocytes, and monocytes for migration to the sites of evolving inflammation. In this study, we reported the
Acknowledgment
This study was supported by the Nation Nature Science Foundation of China (31001120), the Zhejiang Provincial Natural Science Foundation of China (Y3100013) and the Foundation of Zhejiang Educational Committee (Y200908463).
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