Adenovirus-mediated T-bet gene transfers into lymphocyte to induce type-1 T-helper differentiation / 中国病理生理杂志

ABSTRACT

AIM: To construct recombinant adenovirus containing transcription factor T-bet (T-box expressed in T cells),and induce type-1 T-helper differentiation of lymphocytes. METHODS: T-bet gene was cloned from total RNA of lymphocyte stimulated with IFN-? with RT-PCR methods,then subcloned into transfer vector pAdtrack-CMV in BgIII/SaII sites. The new transfer vector pAdtrack-CMV. T-bet was digested with Pme I,subsequently cotransformed into BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The resultant plasmid pAd. T-bet was linearized by Pac I and transfected into 293 cells with liposome LIPOFECTAMINE 2000 for producing Ad.T-bet. The recombined adenovirus Ad.T-bet was identified through RT-PCR and Western blotting methods. Lymphocytes purified from patients suffering from liver cancer was infected with liposome and Ad.T-bet with multiplicity of infection (m.o.i) 5000,and the concentration of IFN-? in culture media was evaluated with ELISA methods. RESULTS: T-bet gene was successfully cloned from lymphocytes and incorporated into recombinant adenovirus Ad.T-bet. Lymphocytes infected with Ad. T-bet constantly and strongly secreted Th1 cytokine IFN-?. CONCLUSION: Recombinant adenovirus Ad.T-bet effectively induces type-1 T-helper differentiation,which is a promising method for restoration of patients' immune reaction against cancer.