Molecular Characteristics Of IRF-2, IRF-3and Functional Analysis Of IRF1of Grass Carp (Ctenopharyngodon Idella)

Interferon regulatory factors (IRFs) are well-known to be crucial for modulating the innate immune responses to viral infections. Although a great progress have get in the researching of fish interferon system in recently years, the specific signal transduction pathway and molecular mechanism are still not very clear. IRF-2and IRF-3gene of grass carp(Ctenopharyngodon idella)(termed CiIRF-2and CiIRF-3, respectively) also were cloned and characterized in the present study. Much attention was payed to the function analyze of the CiIRF-1, IRF-1of grass carp had been reported, in the study. The complete cDNA sequence of CiIRF-2was1809bp in length and comprised a35bp5’-UTR, a793bp3’-UTR and a981bp ORF which encoded a Polypeptide of326amino acids. The putative CiIRF-2was characterized by a conserved N-terminal DNA binding domain(DBD), and included a signature of six conserved tryptophan residues. Phylogenetic relationship analysis revealed that CiIRF-2was highly homologous to the counterparts of other teleosts and mammalians, and nearest to the Danio rerio. While the complete cDNA sequence of CiIRF-3was1809bp. A192bp5’-UTR, a262bp3’-UTR and a1377bp ORF were included in the cDNA. The open reading frame (ORF) of CiIRF-3encoded a Polypeptide of458amino acids, a conserved N-terminal DBD, containing five conserved tryptophan residues, and a conserved C-terminal IRF association domain (IAD) were included. Phylogenetic relationship analysis revealed that CiIRF-3was highly homologous to the counterparts of other higher vertebrates. RT-PCR showed that excepted in drain tissue, CiIRF-3was expressed at a low constitutive level in all tested tissue, but was significantly up-regulated following treatment with Poly Ⅰ:C. Gel mobility shift assays were used to analyze the interaction between the recombinant CiIRF-3and CiIFN promoter sequences in vitro. The results revealed that CiIRF-3could bind to CiIFN promoter with high affinity in vitro. Maybe CiIRF-3acts as an important regulator in the transcription of grass carp IFN gene.CiIRF-1was expressed at a low constitutive level but was significantly up-regulated following stimulation with either Poly Ⅰ:C or recombinant grass carp (C. idella) IFN (rCiIFN) in all6tested tissues by RT-PCR. Gel mobility shift assays were employed to analyze the interaction between the recombinant CiIRF-1and CiIFN/IRF1promoter sequences in vitro. The results revealed that CiIRF-1could bind to CiIFN/IRF1promoter with high affinity in vitro. Subsequently, the recombinant plasmid of pGL3-CiIFNPs and pcDNA3.1-CiIRF-1were constructed and transiently co-transfected into C. idella kidney (CIK) cells or human umbilical vein endothelial cells (HUVEC). The impact of CiIRF-1on CiIFN promoter sequences were measured by luciferase assays. The result showed that the luciferase activity of cell which co-transfected pGL3-CiIFNPs/IRF1-promoter and pcDNA3.1-CiIRF-1was much stronger than of cell only transfected pGL3-CiIFNPs/IRF1-promoter. From all these results we can tell that CiIRF-1acts as a positive regulator in the transcription of grass carp IFN/IRF1gene.