Molecular Cloning, Identification And Characterization Of Immune-relevant Genes In Snakehead Channa Argus

Fish immune system contains innate immune response and acquired immune response. The parenchyma of head kidney is supported by the stroma consisting of reticular cells and fibroblasts. The cells are responsible for the nonspecific and specific immune defenses including lymphocytes, monocytes, plasma cells, granulocytes (two or three types) and macrophages. The immune cells in head kidney are abundant, and may be an important germinal center. In an effort to develop reagents to clarify the immune mechanism involved in snakehead responsive to pathogen infection, and importantly to make a breakthrough in molecular study on fish immune system. A model system for studying fish immune-relevant genes has been established through the mRNA of the stimulated group with poly I:C used as tester and the control group used as driver. A subtractive cDNA library accumulating an enhanced mRNA level of fish immune-relevant genes has been constructed by suppression subtractive technology (SSH). Differential ESTs have been identified through randomly screening colonies from the subtractive cDNA library by both PCR and dot blot. Sequencing analysis revealed that these genes have homology to that in other species. These genes are implicated in signal transduction and modulation, STAT and IRF, antivirus protein, Viperin, ISG15 and ISG12, other important immune genes including Rhamnose binding lectin (RBL), MHC class I, IgH, IgL, HSP70, receptor activated for C protein kinase (RACK), et al.The cDNA sequences of snakehead STAT1, IRF1, Viperin, ISG15 were obtained by RACE-PCR. Together with snakehead IRF1 and known IRF2 and IRF7 molecules, degenerate primers of IRF2 and IRF7 were designed from a conserved region located at the start of the open reading frame (ORF) to specifically amplify IRF2 and IRF7 gene fragments. We performed the cloning cDNA sequences and genomic organization of three IRF genes from the snakehead. The gene organization of IRF-1 and IRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. However, the gene organization of IRF-7 consisting of ten exons and nine introns is different to human IRF-7a gene which has an intron in 5′UTR. Moreover, we detected their distribution in ten tissues, and further demonstrated that these genes are rapidly and transiently induced by poly I:C in spleen and liver. The regulatory sequences of the IRF1, IRF2 and IRF7 5′flanking region have been cloned and sequenced through gene walking, the analysis of snakehead IRF promoters was aim to determine the critical factors that regulate the expression of IRF genes as well as the possible mechanism by which expression of IRF genes are capable of induction by virus and dsRNA. Moreover, the 5′flanking region of STAT1, Viperin and ISG15 were obtained using a genome walking approach. The present study was performed to identify two IFN stimulated genes and to compare the characterization of Viperin and ISG15 promoters in the snakehead.The important characteristic features in the Viperin and ISG15 promoter regions were the presence of interferon stimulating response elements (ISRE) andγ-IFN activation sites (GAS). One conserved site for NF-κB was found in the promoter region of Viperin. This is first reported conservative binding motif for NF-κB in accordance with the consensus sequence (GGGRNNYYCC) among teleost ISG promoters. The characteristics of snakehead STAT1 and IRF7 promoter suggested that mammal intracellular amplifier circuit also exist controlling cellular responsiveness to the IFNs in fish. Subsequently, we discuss the potential roles of these IRFs in regulating immune-related genes and virus- or IFN-mediated signaling pathways. From the lower fish to higher mammals, though IFN and antiviral proteins present a different level of divergence, reflecting to some extent that selection pressures from different pathogens encountered in fish and other organisms because they stay at different environments and have very different life-styles. However, according to our results, not only IFN signal pathway is substantially conserved but also the regulation patterns of IFN regulatory factors and the expression patterns of IFN stimulated gene similar to mammals. In addation, the cDNA sequences of MHC class I, Ig, RBL were obtained by RACE-PCR. Genomic sequences of RBL and its promoter were obtained by genome walking. Expression of STAT1, Viperin, ISG15, MHC class I and RBL genes in tissues were analyzed by RT-PCR. The induced effect of Viperin and ISG15 were detected by the stimulated of poly I:C in liver.In this paper, through SSH and homology cloning, we have cloned, identified and characterized IFN system genes, including STAT1, IRF1, IRF2, IRF7, Viperin, ISG15, and other immune genes. A complex regulatory network implicated in modulating the expressions of fish IFN system genes during virus infection has been preliminary revealed by further studying the structure, expression, induction and evolution of some important genes. The researches of snakehead immune system are essential for understanding immune mechanism and antiviral drug discovery in fish.