Fig. 1. SDS–PAGE analysis of the expressed ESAT-6–MPT64 fusion protein in E. coli. Samples were electrophoresed on 12% SDS–PAGE. Proteins were visualized by Coomassie blue R-250 staining. Lane M, molecular weight markers (kDa); Lane 1, non-induced cell lysate; Lanes 2–4, induced cell lysate at 4 h.

Fig. 2. Western-blotting analysis of the fusion protein. Lanes 1, 3, 5, induced cell lysate; Lanes 2, 4, 6, non-induced cell lysate; blotted with: Lane 1, mouse anti-his monoclonal antibodies; Lane 3, Rabbit anti-ESAT-6 polyclonal antibodies; Lane 5, Rabbit anti-MPT64 polyclonal antibodies.

Fig. 3. SDS–PAGE analysis of purification of the fusion protein. Lane M, molecular weight markers (kDa); Lane 1, E. coli. cell lysate; Lane 2, sample before being loaded onto Ni–NTA column; Lanes 3 and 4 wash with 8 M urea wash buffer pH 6.0; Lane 5, cytosol fraction of the E. coli transformant sonicate; Lane 6, insoluble protein fraction obtained from E. coli transformant sonicate; Lanes 7 and 8, wash with 8 M urea wash buffer pH 5.3; Lanes 9 and 10, wash with 8 M urea elution buffer pH 4.0.

Fig. 4. The kinetic study of antibody responses of immunized mice. Antibody titers against the antigens were determined by ELISA assays from immunized mice (n = 10) at 1 week intervals. Indicated the time of the second and third immunizations. Indicated the time of bacterial challenge. The experiment was performed twice with similar results.

Fig. 5. Proliferations of splenic lymphocytes of immunized mice. Two weeks after the final immunization, splenic lymphocytes (5 × 10⁵ cells/well) from sacrificed C57BL/6 J mice (n = 10) were pooled and stimulated with 5 μg/ml mycobacterium CFPs for 72 h. Values and bars represent the mean of stimulation index of triplicate values ± standard deviation. ^**Significantly different from MPT64 group (P < 0.01).

Fig. 6. IFN-γ and IL-12 releases from splenic lymphocytes of immunized mice. Splenic lymphocytes (5 × 10⁶ cells/well) from sacrificed mice were pooled and stimulated with 5 μg/ml proteins for 72 h. (A) IFN-γ productions in groups were assayed. (B) IL-12 (p40) productions in groups were assayed. Values and bars represent the mean of released cytokine concentrations of triplicate values ± standard deviation. ^**Significantly different from MPT64 group (P < 0.01).

Purification and refolding steps of ESAT-6–MPT64 fusion protein from culture of E.coli

Bacterial counts in the lungs and spleens of mice immunized with proteins and challenged by M. tuberculosis H37Rv