PRELIMINARY INVESTIGATION ON CRUCIAN CARP INTERFERON REGULATORY FACTOR 1 ROLE IN FISH INTERFERON SYSTEM

More attention has been paid to fish interferon(IFN) system since it played a key role in fish innate immune system.The interferon regulatory factor(IRF) family comprises transcription factors that regulate the expression of interferon and IFN-stimulated genes(ISGs) by binding to characteristic elements in their promoters.Among IRFs,at least four members,IRF1,IRF3,IRF5 and IRF7,have been implicated as direct transducers of virus-mediated IFN signaling.IRF1 was the first IRF family member known to activate the IFNβ gene,and found to be constitutively expressed in most cell types.In our laboratory,a cDNA library of CAB cells(Blastulae embryonic cells of crucian carp) has been established,which is an ideal cell model system for studying fish antiviral-relevant genes,and the fulllength cDNA of crucian carp IRF1(CaIRF1) was cloned from the library.Furthermore,subcellular localization and inductive expression of CaIRF1 were also characterized in the previous study.In order to further investigate the role of CaIRF1 in fish innate immune system,the stably transfected cells were obtained with CaIRF1 overexpression in the study.Firstly,the cDNA fragment containing C-terminal 165 amino acids of CaIRF1 was cloned and integrated into a prokaryotic expression vector pET32a,and then a recombinant protein with the expected size was expressed under the induction of IPTG and purified according to the protocol of the His.Bind Purification Kit.The anti-CaIRF1 polyclonal antibody was prepared by immunizing rabbit with the purified recombinant protein and subsequently used to detect the stably transfected cells in western blotting analysis.Secondly,we cloned CaIRF1 gene with in-frame restriction sites into a eukaryotic expression vector pcDNA3.1,which is under the control of a strong cytomegalovirus immediate-early gene promoter.Then,the recombinant eukaryotic expression vector was transfected into CAB cells,and the stably transfected cells were selected with G418 for a month.After that,the stably transfected cells were detected to confirm the CaIRF1 overexpression by real-time PCR and western blotting analysis.Real-time PCR analysis showed expression of CaIRF1 was obviously higher in mRNA level in the stably transfected cells than that in the control cells;Moreover,the data also revealed that mRNA level of IFN gene was up-regulated in the stably transfected cells.Correspondingly,expression of CaIRF1 was also increased in protein level in the stably transfected cells as compared with the control cells by western blotting analysis;to verify the specificity of the anti-CaIRF1 polyclonal antibody,the antiserum was pre-adsorbed with the purified recombinant CaIRF1-C/His peptide for 16h at 4℃.As a result,the pre-adsorbed antiserum could not recognize the corresponding peptide in the subsequent western blotting.So,the results from real-time PCR and western blotting analysis confirmed that CaIRF1 had been overexpressed in the stably transfected cells.To study effect of CaIRF1 overexpression on expression levels of other genes in interferon system,the stably transfected cells were induced with poly I:C,and the two genes,STAT1 and IFI58 were detected by real-time PCR.The results displayed that the expression were obviously up-regulated in mRNA levels.In conclusion,our data indicates that CaIRF1 may have the same function as mammalian IRF1,which mediates the antiviral state in cells by regulating the expression of IFN and ISG genes.Therefore,further studies will be needed to understand the importance of CaIRF1 in fish interferon system.