Molecular Cloning And Characterization Of IRF5 From Black Carp

Black carp is an important freshwater economic fish species in China.However,pathogenic microorganisms present in nature and culture environment pose a serious threat to it's culture industry,such as viruses.In this study,the black carp(Mylopharyngodon piceus)IRF5(bcIRF5),a member of interferon regulated factor 5(IRF 5),was used as the research object to explore the innate immunity process.In this study,the bcIRF5 gene was cloned and it was recombinantly expressed and functionally analyzed.The following results were obtained: 1.Compared with other animal IRF5 homologs,bcIRF5 is highly conserved in sequence and structure.The amino acid sequence is very similar to the amino acid sequence of grass carp and zebrafish IRF5,which are 99.6% and 93.6% respectively;and its two important domains DNA binding domain(DNA banding domain(DBD)and IRF-associated domains(IAD)are highly conserved;2.Real-time quantitative PCR showed that spring viremia of carp virus(SVCV)and grass carp reovirus(GCRV)could affect the expression of bcIRF5 in different organs,similarly,two simulated stimuli(Poly(I:C)or LPS)and SVCV or GCRV also affect the mRNA expression level of bcIRF5 in the host cell,MPK;3.In EPC or HEK293 T cells,the size of the bcIRF5 expressed by recombinant vectors is about 65 k Da by western blotting;4.bcIRF5 wascharacterized as a predominantly cytosolic protein by immunofluorescent staining in EPC cells and MPK cells and showed little IFN promoter-inducing ability in reporter assay;5.The direct association between bcIRF5 and black carp TBK1(bcTBK1)were identified through co-immunoprecipitation assay,and co-expressed bcIRF5 in EPC cells suppressed bcTBK1-mediated IFN promoter transcription in reporter assay.Surprisingly,the titer of grass carp reovirus(GCRV)in the media of EPC cells co-expressing bcIRF5 and bcTBK1 was obviously lower than that of EPC cells expressing bcTBK1 alone.It was interesting that expression of bcIRF5 and/or bcTBK1 in EPC cells showed little effect on cell growth;however,the survival ratio of EPC cells co-expressing bc TBK1 and bcIRF5 post GCRV infection was much lower than that of EPC cells expressing bcIRF5 or bcTBK1 alone.In short,these results indicate that bcIRF5 involves in the antiviral innate immune of black carp.On the one hand,bcIRF5 negatively regulates bcTBK1-mediated IFN signaling,which may be related to suppressing the occurrence of hyperimmune;on the other hand,it correlates with bcTBK1 and triggers cell death to inhibit the virus replication during the innate immune activation.