Identification Of The Interaction Between TAB2 And TAK1 Proteins From Ctenopharyngodon Idella And Effect Of Their Interaction On NF-κB Pathway

Transforming growth factor-beta activated kinase 1(TAK1)binding proteins(TABs)family include three members:TAB1,TAB2 and TAB3.In mammals,it has been confirmed that TAB2 interact with TAK1 can activate the nuclear factor kappa-B(NF-κB)pathway which play an important role in antibacterial immunity through regulating the expression of cytokines and antimicrobial peptides.However,there are few studies in fish about the interaction between TAB2 and TAK1 and effect of their interaction on the antibacterial immunity regulation via activating the NF-κB pathway,nor in grass carp.Our previous studies shown that the transcription of Ctenopharyngodon idella(C.idella)TAK1(CiTAK1)and TAB1(CiTAB1)in tissues were upregulated significantly after the Vibrio mimicus(V.mimicus)infection and its vaccine immunity.And confirmed that CiTAK1could interact with CiTAB1.On these bases,CiTAB2 was taken as the research object in this study.First,detecting the transcription of CiTAB2 gene in grass carp tissues after V.mimicus infection or in CIK cells after OVeips immunostimulation.Then,identifying the interaction between CiTAK1 and CiTAB2.Finally,exploring the effect of their interaction on NF-κB pathway.All these results could enrich the understanding of fish antibacterial immunity which regulated by TAK1-TAB2/NF-κB pathway and provide some new strategies for prevention and treatment on the disease which causing by V.mimicus.The primary research contents and results were summarized as follows.1.CiTAB2 response to V.mimicus infection and immunostimulationFirstly,V.mimicus(1×10~8 CFU/Tail)was used to infect the grass carp,and then detecting the transcription level of CiTAB2 in liver,spleen and head kidney by RT-q PCR before and after infection.After that,the V.mimicus vaccine antige(OVeips)with different concentrations were used to immunostimulate the C.idella kidney(CIK)cells,and RT-q PCR was used to detect the transcription level of CiTAB2 in CIK cells before and after the immunostimulation.The results shows that the transcription level of CiTAB2 in immune tissue was significantly upregulated after V.mimicus infection,and the transcription level of CiTAB2 in CIK cells was also significantly changed after OVeips immunostimulation,and this changes were relative to the OVeips’concentration and immune time.Indicated that CiTAB2 involved in the immune process which against V.mimicus infection in grass carp.2.Identification of the interaction between CiTAB2 with CiTAK1Firstly,the recombinant eukaryotic expression plasmids p CMV-Myc-CiTAB2 was successfully constructed.Then,p CMV-Myc-CiTAB2 and p EGFP-C1-CiTAK1 were co-transfected in Human embryonic kidney(HEK293T)cells.Later,the harvested cell lysate was detected by Western blotting with Anti-GFP and Anti-Myc after co-immunoprecipitation(Co-IP)with Anti-Myc immunomagnetic beads.Finally,pm Cherry-C1-CiTAB2 or/and p EGFP-C1-CiTAK1 were transfected into CIK cells or HEK293T cells.After paraformaldehyde fixture and DAPI staining,the subcellular localization of the fusion proteins was observed under fluorescence microscope.Results shows that Myc-CiTAB2 in the cell lysates which co-transfected with pm Cherry-C1-CiTAB2 and p EGFP-C1-CiTAK1was pulled down by Co-IP,and Myc-CiTAB2 or EGFP-CiTAK1 can be detected by Anti-EGFP or Anti-Myc.And found that CiTAB2 could express in cytoplasm and co-localize with CiTAK1.Indicated that CiTAB2 can interact with CiTAK1 in cells.3.Effect of the interaction between CiTAB2 and CiTAK1 on NF-κB pathwayFirstly,the eukaryotic overexpression vector p EGFP-C1-CiTAB2 was successfully constructed.Then,p EGFP-C1-CiTAB2 and p EGFP-C1-CiTAK1 were co-transfected into CIK cells,and the expression of them were detected by Western blotting.Finally,detecting the transcriptional level of key genes in NF-κB pathway and downstream genes in CIK cells by RT-q PCR after transfected with p EGFP-C1-CiTAB2 and/or p EGFP-C1-CiTAK1.The results shows that both EGFP-CiTAB2 and EGFP-CiTAK1 could express in CIK cells,and their overexpression could significantly upregulate the transcription levels of key genes in NF-κB pathway,downstream cytokines as well as antimicrobial peptide genes.Indicated that the interaction between CiTAB2 and CiTAK1 have significantly affection on NF-κB pathway.In conclusion,V.mimicus infection and OVeips immunostimulation can significantly promote the transcription of CiTAB2 gene.CiTAB2 can interact with CiTAK1 in cells,and their interaction can significantly affect the transcription levels of key genes in NF-κB pathway,downstream cytokines and antimicrobial peptide genes.All these results could enrich the understanding of fish antibacterial immunity which regulated by TAK1-TAB2/NF-κB pathway and provide some new strategies for prevention and treatment on the disease which causing by V.mimicus.