Molecular cloning and functional characterization of interferon regulatory factor 7 of the barbel chub, Squaliobarbus curriculus

Abstract

The interferon regulatory factor 7 (IRF7) is a critical regulator of type-I interferon-dependent immune reaction that defense against virus. To investigate the antiviral function of IRF7 of barbel chub Squaliobarbus curriculus (ScIRF7), the cDNA of ScIRF7 was cloned and characterized. The full length cDNA of ScIRF7 was 1870 bp, consisted of 41 bp 5′-UTR, 560 bp 3′-UTR and a 1269 bp open reading frame (ORF). The ORF encoded 423 amino acids with a molecular weight of 49.426 KDa and a theoretical isoelectric point of 5.71. The putative ScIRF7 protein possesses typical domains of IRF family including a conserved N-terminal DBD-binding domain (DBD), a C-terminal IRF association domain and a serine-rich domain. In the DBD, four tryptophans were found to be highly conserved among all species, whilst in another conserved tryptophan site of mammals, the corresponding amino acids were methionine for fishes. The expression level of ScIRF7 was highest in the spleen and lowest in the liver. The expression level of IFN-β was highest in the gill and lowest in the liver. After GCRV infection, expression levels changes of ScIRF7 showed an overall tendency of firstly up-regulation and then down-regulation in the spleen and the gill; and expression levels of ScIRF7 in peripheral blood lymphocyte at 24 h post-infection was highest among all time points. In pEGFP-ScIRF7 overexpressing cells, the mRNA level of ScIRF7 was firstly up-regulation and then down-regulation; and the expression of IFN-β was significantly up-regulated at 12 h post-infection than that of control group (P < 0.05), which was significantly higher than those in pEGFP-N1 overexpressing cells. The results indicated that ScIRF7 may play a key role in immune responses of barbel chub Squaliobarbus curriculus against GCRV and may also functions in the Ctenopharyngodon idellus kidney cells.