Study On The Clone And Biological Function Of T-bet Gene

T-bet gene (T-box expressed in T cells) includes an open readingframe of 1607bp, which encodes 530 amino acid, mainly expressed inlung, spleen and thymus. The distribution of the tissue instructed thatT-bet is specific to lymphocytes. T-bet as a specific transcription factor ofThl cells can induce IFN-γand IL-12Rβ2 expression and meantimesuppress IL-4 secretion.Objective:(1) Clone and identify human T-bet gene.(2) Express T-bet protein in E.coli, immunize Balb/c mice withpurified T-bet protein and prepare monoclonal antibody against T-bet.(3) Electroporate THP-1 cells with pT-bet (T-bet gene wasconstructed into pIRES₂-eGFP plasmid) and sceen out single clone withG418.(4) Construct trans-membrane vector to express T-bet protein withTAT and transfect THP-1 cells to test the modulation effect on T cells.Methods:(1) PBMCs were isolated from donor peripheral blood, the totalRNA was extracted and transcribed into cDNA. The T-bet gene wasamplified by PCR with specific primers and constructed intopGEM-Teasy vector. The sequence of the T-bet gene was detected by single and double restriction enzyme digestion as well as sequencinganalysis.(2) T-bet gene was constructed into pQE30 vector and expressed inE.coli M15 to get T-bet fusion protein; The fusion protein was purified byIMAC column of Bio-Rad company; The purified protein was used toimmunize Balb/c mice, then splenic cells from immunized mice werecollected to fuse with SP2/0 cells and culture in HAT medium to gethybridoma cell line which secrect T-bet antibody. T-bet gene wasconstructed into pIRES₂-eGFP, and the recombined plasmid waselectroporated into THP-1 cells (pIRES as negative control); The cellline of THP-1 expressed T-bet was screened, which was used to test themonoclonal antibody by Western-blot.(3) pQE30 vector was reconstructed via inserted TAT sequence,which owned trans-membrane function. T-bet was inserted into the newvector and expressed in E.coli M15, the T-bet protein withtrans-membrane function was succeed; The purified T-bet protein solutionwas added into THP-1 culture medium, the trans-membrane process andefficiency was monitored. Furthermore, the treated THP-1 cells wereco-cultured with CD4⁺T cells. This kind of model was used to observe thefunction of T-bet in modulation of Th1/Th2 balance. (1) The full length of T-bet gene with open reading frame was clonedsuccessfully from human PBMC.(2) The monoclonal antibody against human T-bet protein wasgained, which was approved with high reactivity and specificity byELISA and Western-blot, respectively.(3) The T-bet protein with trans-membrane function was produced,which was used to transfect THP-1 cells and the function to induce Th1cells was observed.Conclusion:In short, T-bet gene was cloned and expressed in E.coli systemeffectively; The purified protein acted as antigen used to immunize miceand monoclonal antibody against T-bet was produced using hybridomatechnique; The gene- and protein-transfection methods were used totransfect T-bet into THP-1 cells and T-bet function towards regulation ofTh1/Th2 cells was studied. All of these work make an initial foundationfor further research the function of T-bet.