Molecular Cloning And Functional Study Of IRF9 From Black Carp

Interferon regulatory factor 9(IRF9)is an important transcriptional regulatory factor in JAK-STAT signaling.In this study,the coding sequence(CDS)of the IRF9 of black carp was cloned,and its antiviral function was initially explored.The CDS sequence of bc IRF9 gene contains 1287 nucleotides,which can be translated into 428 amino acids;ExPASy software predicts that the molecular weight of bcIRF9 is48.31 k Da and the isoelectric point is 7.98.Secondly,the structure prediction revealed that bcIRF9 is conserved in structural evolution and mainly contains the N-terminal DNA-binding domain(located at 1-120 aa,DBD),the C-terminal IRF-association domain(located at 216-428 aa,IAD),and the intermediate linker domain(located at 121-215 aa,LD).Real-time quantitative PCR showed that the level of bcIRF9 mRNA in the kidney cells of the host increased with the stimulation of LPS,poly(I:C),and GCRV.Immunoblot experiments showed that the eukaryotic expression vector of bcIRF9 in HEK293 T cells and EPC cells can be successfully expressed,and the band size is about 54 KDa.Immunofluorescence experiments identified that bc IRF9 is concentrated in the nucleus.We constructed the domain deletion mutant of bcIRF9 to explore the impact of different domains on its location and function,the result first showed that its linker domain(LD)can affect its localization.The double luciferase reporter gene experiment found that bcIRF9 can up-regulate the interferon-stimulated response element(ISRE)promoterin a dose-dependent manner.Second,its DBD and LD are essential for its ability of up-regulating the ISRE promoter.Our research group has reported that two subtypes of bcSTAT1(bcSTAT1a and bcSTAT1b)can play an important role in host antiviral immunity.However,little is known about the role of STAT1 and IRF9 in teleost fish.Luciferase reporter assay found that when bcIRF9 and bcSTAT1 a or bcSTAT1 b are overexpressed in EPC,bcSTAT1 a can up-regulate the ability of bcIRF9 to induce ISRE,but bcSTAT1 b does the opposite.The plaque assay data demonstrated that EPC cells expressing both bcSTAT1 a and bcIRF9 have significantly enhanced antiviral ability than EPC cells expressing bcIRF9 alone.However,EPC cells expressing both bcSTAT1 b and bc IRF9 have significantly reduced antiviral capabilities than EPC cells expressing bcIRF9 alone.In summary,this study demonstrated that bcIRF9 plays an important role in the innate defense against GCRV,in which two STAT1 proteins function differently.