Molecular Cloning And Functional Study Of TAK1 From Black Carp

Innate immunity is essential for teleost fish against pathogenic microbial infections.Transforming growth factor-?-activated kinase 1(TAK1)is a member of the serine/threonine protein kinase family and plays an important role in NF-?B and AP-1 inflammatory signaling pathways however the function of TAK1 in antiviral innate immune IRF/IFN pathways is unclear.In this paper,the open reading frame(ORF)of the TAK1 gene of black carp(Mylopharyngodon piceus)was cloned and its function in the antiviral innate immune response of host cells was studied.The open reading frame of the bcTAK1 gene contains 1626 nucleotides encoding 541 amino acids;ExPASy software predicts that bcTAK1 has a molecular weight of 75 Kda and an isoelectric point of 7.55.bcTAK1 has the highest homology with grass carp TAK1(up to 98.5%),and has a higher homology with mammals TAK1.It is conserved in structural evolution and mainly consists of N-terminal serine/threonine protein kinase domain(S/TKc,12-260aa)and C-terminal coiled-up coil domain(465-528aa).Western blotting showed that the bcTAK1 gene was efficiently expressed in both HEK293 T cells and EPC cells,but its migration band suggested that bcTAK1 has glycosylation modification.Immunofluorescence experiments showed that bcTAK1 was distributed in the cytoplasmic region and had a spot-like distribution.Dual luciferase reporter gene analysis indicated that bcTAK1 could down-regulate the transcription of bcIFNa,DrIFN?1,DrIFN?2,DrIFN?3,and fathead minnow IFN.When both bcTAK1 and black carp interferon regulatory factor 7(bcIRF7)were overexpressed in EPC cells,bcTAK1 significantly up-regulated the ability of bcIRF7 to induce IFN production.The results of virus titer assay showed that EPC cells expressing bcTAK1 alone were not affected by the ability of grass carp reovirus(GCRV)and sputum viremia virus(SVCV)compared with control cells;The antiviral ability of bcIRF7 overexpressed EPC cells was significantly improved.However,EPC cells expressing both bcTAK1 and bcIRF7 have significantly enhanced antiviral ability than EPC cells expressing bcIRF7 alone.In summary,this paper reveals that bcTAK1 has the role of positively regulating bcIRF7-induced IFN production and positively regulating bcIRF7-mediated antiviral activity in the anti-viral innate immune response of black carp.This was first discovered in vertebrates and even humans.