PREL IM INARY INVESTIGATI ON ON CRUC I AN 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 immunesystem. The interferon regulatory factor (IRF) family comprises transcription factors that regulate the expression of interfer on and IFN stimulated genes (ISGs) by binding to characteristic elements in their promoters. Among IRFs, at least fourmembers, 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 IFN β gene, and found to be constitutively expressed in most celltypes. 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 full length cDNA of crucian carpIRF1 (CaIRF1) was cloned from the library. Furthermore, subcellular localization and inductive expression of CaIRF.were also characterized in the previous study. In order to further investigate the role of CaIRF1 in fish innate immune system, the stably transfected cellswere obtained with CaIRF1 overexpression in the study. Firstly, the cDNA fragment containing Cterminal 165 amino acids of Ca IRF1 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 theprotocol of the His. Bind Purification Kit. The anti CaIRF1 polyclonal antibodywas prepared by immunizing rabbitwith thepurified recombinant protein and subsequently used to detect the stably transfected cells in western blotting analysis.Secondly,we cloned CaIRF1 gene with inframe restriction sites into a eukaryotic expression vector pcDNA311,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 cellswere selected with G418 for a month. Afterthat, the stably transfected cellswere detected to confirm the CaIRF1 over expression by realtime PCR and western blottinganalysis. Realtime 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 upregulated inthe stably transfected cells. Correspondingly, expression of CaIRF1 was also increased in protein level in the stably transfected cells as compared with the control cells bywestern blotting analysis; to verify the specificity of the anti CaIRF1 polyclonal antibody, the antiserum was preadsorbed with the purified recombinant CaIRF12 C/His peptide for 16h at 4℃. As aresult, the pre2adsorbed antiserum could not recognize the corresponding peptide in the subsequentwestern blotting. So, theresults from real2time 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 stablytransfected cellswere induced with poly I: C, and the two genes, STAT1 and IFI58 were detected by realtime PCR. The results displayed that the expression were obviously upregulated in mRNA levels1In conclusion, our data indicates that Ca IRF1 may have the same function asmammalian IRF1,which mediates the antiviral state in cells by regulating the expression of IFN and ISG genes1Therefore, further studieswill be needed to understand the importance of CaIRF1 in fish interferon system.