Study On The Biological Function Of Type ? Interferon IFNe2 In Chinese Sturgeon(Acipenser Sinensis)

Type ? interferon(IFN)is a polypeptide secreted by a pathogen-infected cell,which has antiviral,antibacterial and antitumor activities,and promotes immune response regulation.Therefore,understanding the function of IFNe,which is highly expressed in sick Chinese sturgeon Acipenser sinensis,is beneficial to enhance the protection of Chinese sturgeon and to deepen the understanding of the mechanism of immune evolution.In this study,we performed a preliminary study on the in vitro recombination of Chinese sturgeon IFNe2 protein.In this study,we cloned the Chinese sturgeon IFNe2 coding region which removed the signal peptide into the prokaryotic expression vector of pET-32 a and found that the Chinese sturgeon IFNe2 was mainly expressed as soluble protein in E.coli,and purified by nickel affinity chromatography.In order to further study the function of IFNe2,we cloned the sequence between the IRF3 and IRF7 DBD(DNA-binding domain)domain and the IAD(IRF association domain)domain into the pET-32 a vector.It was found that both IRF3 and IRF7 can be expressed as soluble proteins,which can be purified for polyclonal antibody preparation.Higher titers of IRF3 and IRF7 polyclonal antibodies were obtained after four times of immunization,with IRF3 antibody titers exceeding1:729000 and IRF7 antibody titers exceeding 1:81000.In addition,the specificity of IRF3 and IRF7 polyclonal antibody was detected by Western Blot,which found that both polyclonal antibodies have strong specificity.In the Chinese sturgeon,we have studied the temporal and spatial expression of IFNe2,but its function is not clear.In the present study,we examined the antiviral effects of IFNe2 and investigated the effect of IFNe2 on IFN signaling.The results showed that the addition of recombinant Chinese sturgeon IFNe2 protein at a final concentration of 1 ?g/mL induce the expression of Mx,PKR and Viperin in EPC(epithelioma papulosum cyprini)cells,indicated that the recombinant IFNe2 activated the antiviral activity of EPC cells.Besides,it was found that the expression of SVCV(spring viraemia of carp virus)G protein,N protein and P protein in EPC cells was decreased by adding recombinant IFNe2 protein,and the pathological effect was also reduced by adding recombinant IFNe2 protein.This result indicates that Chinese sturgeon IFNe2 possesses antiviral activity.Recombinant Chinese sturgeon IFNe2 protein can also affect the expression of genes on the IFN signaling pathway.The expression of interferon-stimulated genes such as Mx,PKR,Viperin and ADAR4 was induced by the adding 1 ?g/mL recombinant IFNe2 protein to the Chinese sturgeon fincell line,especially the expression of Mx and Viperin was up-regulated significantly.The expression of ADAR1-1 and ADAR1-2 could not be induced by recombinant IFNe2.In addition,recombinant IFNe2 could induce the expression of IRFs.The expression of IRF1 and IRF2 were up-regulated at 3 h and 6 h after induction,however,the expression of IRF3 and IRF7 were continuously up-regulated to 24 h and induced phosphorylation of IRF7.What's more,the expression of IFNe1 and IFNe3 in the cells was significantly up-regulated at 3 h,and then returned to normal levels when adding 1?g/mL recombinant IFNe2 protein to stimulate Chinese sturgeon fin cell line,while the expression of itself was down-regulated at 6 h,followed by a significant up-regulation at 12 h.These results indicate that IFNe2 may expand the immune response of IFN by inducing phosphorylation of IRF7.IRF is a transcription medium for IFN signaling pathways induced by viruses and bacteria,and plays an important role in many cellular processes,such as resistance to virus invasion.IRF plays an important role in the transcription of IFN.IRF plays an important role in the transcription of IFN.In order to understand the regulation of IFNe2 transcription by Chinese Sturgeon IRF,we studied the structure and expression pattern of IRF1,IRF3,IRF4,IRF5 and IRF8 in Dabry's Sturgeon(Acipenser sinensis).Except for IRF1,all IRFs consist of 8 exons and 7 introns.Furthermore,in the predicted protein sequence,the DBD domain has the highest identity with the spotted gar(Lepisosteus oculatus).Regarding the expression patterns,five IRF genes were found to be constitutively expressed in all tissues examined,and was significantly higher in blood,kidney,intestine,and spleen.Following Aeromonas hydrophila infection the expression of IRF1 and IRF3 were up-regulated in the spleen and caudal kidney,while both the IRF5 and IRF8 genes were down-regulated in caudal kidney.In addition,IRF4 was significantly up-regulated at 3 h post-infection by A.hydrophila in the spleen and caudal kidney.These results suggest that IRF1,IRF3 and IRF4 may play a crucial role in bacterial-induced IFN transcription.