The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides variableefficacy against adult pulmonary tuberculosis (TB).Recombinant BCG,expressing either immunodominantantigens or Th1 cytokines,is a promising strategy for developing a new TB vaccine.However,not much isknown about whether the introduction of cytokine and specific antigen genes concurrently into the BCGstrain could improve the immunogenicity of BCG.In this study,a recombinant BCG strain (rBCG) expressingthe fusion protein human interleukin (IL)-2 and ESAT-6 (early secreted antigenic target-6 kDa) antigen ofMycobacterium tuberculosis was constructed.Six weeks after BALB/c mice (H-2~d) were immunizedwith 10~6 colony forming units (CFUs) BCG or rBCG,splenocyte proliferation was determined with MTT[3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay,IL-4 and interferon (IFN)-γproducedby splenocytes were tested by enzyme linked immunosorbent assay (ELISA,) and the cytotoxicity ofsplenocytes from immunized mice to P815 cells (H-2~d) expressing ESAT-6 protein was measured usingCytoTox 96 Non-Radioactive Cytotoxicity Assay.Compared with native BCG-vaccinated mice,rBCG inducedstronger Th1 responses that were confirmed by high lymphoproliferative responses and IFN-γproductionto culture filtrate protein (CFP) or ESAT-6 protein.Moreover,rBCG induced significant enhanced CTLresponses against P815-ESAT-6 cells.Results from rBCG-immunized mice demonstrated that introducingthe il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6.Further investigationis needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacyagainst TB. The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides recombinant efficacy against adult pulmonary tuberculosis (TB). Resulting BCG, expressing either immunodominant antigens or Th1 cytokines, is a promising strategy for developing a new TB vaccine. the introduction of cytokine and specific antigen genes concurrently into the BCG strain could improve the immunogenicity of BCG. this study, a recombinant BCG strain (rBCG) expressing the fusion protein human interleukin (IL) -2 and ESAT-6 (early secreted antigenic target- 6 kDa) antigen of Mycobacterium tuberculosis was constructed. Six weeks later BALB / c mice (H-2 ~ d) were immunized with 10-6 colony forming units (CFUs) BCG or rBCG, splenocyte proliferation was determined with MTT [3- 5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide] assay, IL-4 and interferon (IFN) -γproducedby splenocytes were tested by enzyme linked immunosorbent assay (ELISA) and the cytotoxicity ofplenocytes fr om immunized mice to P815 cells (H-2 ~ d) expressing ESAT-6 protein was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay. Compared with native BCG-vaccinated mice, rBCG induced hypertensive Th1 responses that were confirmed by high lymphoproliferative responses and IFN- γproductionto culture filtrate protein (CFP) or ESAT-6 protein. Moreover, rBCG induced significant enhanced CTLresponses against P815-ESAT-6 cells. Results from rBCG-immunized mice were tested that introduces the il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6.Further investigation necessary needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacy on stomach TB