Transcriptional Expression Patterns Of Grass Carp(Ctenopharyngodon Idella) Immune-related Factor TAB1 And Verification Of The Interaction Between TAB1 With TAK1

When two or more proteins bind non-covalently to form a protein complex,it is termed a protein–protein interaction(PPI).PPI is an important part of the biochemical reaction network in cells.Therefore,PPI study can not only reveal the structure and function of protein at molecular level,but provide a new approach to study pathogenic mechanism and animal epidemic disease prevention and control.Transforming growth factor-?activated kinase-1(TAK1)is a member of the serine/threonine protein kinase family,and TAK1 bingding protein(TAB1)is a highly conserved immune-related protein which widely distribute in invertebrate and vertebrate cells.It has been confirmed that the interaction between mammalian TAB1with TAK1 participate in regulating some cell signaling pathways.Therefore,mammalian TAB1 protein plays an important role in cell differentiation,apoptosis and innate immune response.Compared with mammals,the study on the interaction between TAB1 and TAK1 protein in fish is relative later,only a study has focused on the interaction between TAB1 and TAK1 in large yellow croaker to date.In our previous study,a targeted DNA vaccine of Vibrio mimicus could significantly enhance the intestinal mucosal immune response,and the CiTAB1 gene in intestine were significantly upregulated and enriched in multiple innate immune signaling pathways.But,the relationship between the protein CiTAB1 and CiTAK1 is still unclear.Accordingly,CiTAB1 was used as a research object in the study.The transcriptional expression patterns of CiTAB1 gene in different tissues of healthy grass carp and C.Idella kidney(CIK)cells before and after immune stimulation were analyzed.And the subcellular localization of CiTAB1 protein and its co-localization with CiTAK1protein were determined.On the basis,immunoprecipitation and pull-down were performed to verify the interaction between CiTAB1 and CiTAK1.The main research work and achievements were summarized as follows:1)Transcriptional expression pattern analysis of CiTAB1 gene.Here,the total RNA in healthy grass carp tissues was extracted by using TRNzol-A~+total RNA extracting reagent,and then being reverse transcribed into cDNA.The transcriptional expression profiles of CiTAB1 gene in nine different tissues were firstly detected by RT-qPCR using cDNA as a templet,and we found that CiTAB1 gene was ubiquitous in all examined tissues.Subsequently,lipopolysaccharide,tumor necrosis factor-?and vaccine antigen of Vibrio mimicus(named OVepis)in different concentrations were used to stimulated CIK cells.The temporal transcriptional expression levels of CiTAB1 gene in CIK cells before and after stimulation were detected by RT-qPCR,either.The results showed that CiTAB1 protein was an immune-related protein,and all tested immunostimulants could significantly upregulate the transcription of CiTAK1 gene,but the up-regulated levels varied according to the different immunostimulants and their concentrations.2)Identification of subcellular localization of CiTAB1 protein and its co-localization with CiTAK1 protein.Here,the subcellular localization of CiTAB1protein was firstly predicted using online analysis software.A eukaryotic expression recombinant plasmids with fluorescence labels(pmCherry-C1-CiTAB1)was then constructed.Finally,the resulting recombinant plasmid was transfected into CIK and HEK293T cells or co-transfted with p EGFP-C1-CiTAK1 into HEK293T cells.After transfection,cells were fixed with polyformaldehyde,followed by nucleus stain with DAPI and fluorescence observation under a fluorescence microscopy(400×).The results were shown below:(i)CiTAB1 protein might be distributed in the nucleus predicted by online software;(ii)the CIK and HEK293T cells transfected with pmCherry-C1-CiTAB1 alone emitted red fluorescence in the cytoplasm and nuclei;(iii)the HEK293T cells co-transfected with p EGFP-C1-CiTAK1 and pmCherry-C1-CiTAB1,green fluorescence(EGFP-CiTAK1)overlapped with red fluorescence(m Cherry-CiTAB1)to form a yellow fluorescence in the cytoplasm.These results suggested that CiTAB1 was a protein that distributed throughout the entire cells,and can co-localize with CiTAK1 in the cytoplasm.3)Verification of CiTAB1 and CiTAK1 interaction.Here,the pCMV-myc-CiTAK1 and pmCherry-C1-CiTAB1 or targeted protein(pCMV-Myc,pmCherry-C1)were co-transfected into HEK293T cells.The cell lysates were immunoprecipitated with anti-myc immunomagnetic bead and then analyzed by western blotting.The results showed that myc-CiTAK1 could be detected from IP lysate of transfection pCMV-Myc-CiTAK1 and pmCherry-C1-CiTAB1 or pmCherry-C1 by using anti-Myc,mCherry-C1-CiTAB1 could be detected from IP lysate of transfection pCMV-Myc-CiTAK1 and pmCherry-C1-CiTAB1 by using anti-mCherry,which indicated that the protein CiTAB1 could interact with CiTAK1.In order to further verify whether the above two proteins could directly interact in vitro,His-CiTAB1 fusion protein was used as bait protein and CiTAK1 was used as prey protein,and then analyzed by western blotting.The results showed that fusion protein His-CiTAB1 and His target protein could be detected from the mixture of teansfected pCMV-Myc-CiTAK1 lystates and the fusion protein His-CiTAB1 or the His target protein by using anti-His monoclonal antibody;Myc-CiTAK1 could be detected from the mixture of teansfected pCMV-Myc-CiTAK1 lystates and recombinant protein using anti-Myc monoclonal antibody,but could not be detect in the mixture of teansfected pCMV-Myc-CiTAK1 lystates and His tag protein.These results suggested that CiTAB1could directly interact with CiTAK1 proteins in vitro.In summary,CiTAB1 gene was ubiquitously transcriptional expression in all examined tissues,which could be significantly upregulated by all tested immunostimulants.CiTAB1 protein was distributed in both cytoplasm and nucleus,and co-localized with CiTAK1 protein in cytoplasm.There is a direct interaction between CiTAB1 and CiTAK1 proteins in vitro.