[Preparation of TAT-Ag85B protein vaccine and evaluation of its anti-Myobacterium tuberculosis effect].

OBJECTIVE

To obtain a new Ag85B protein fused with protein transduction domain (PTD) produced by a HIV-trans-activating transduction domain (TAT-PTD) expression system and investigate its protective effect against Myobacterium tuberculosis as a subunit vaccine.

METHODS

The pET28a-Ag85B and pET28a-TAT-Ag85B plasmids were established and transformed into E.coli BL21(DE3) strains for recombinant protein expression and purification. Then three groups of BALB/c mice were subcutaneously vaccinated three times with Ag85B protein, TAT-Ag85B protein and PBS, respectively. One week after the last immunization, 5 mice in each group were sacrificed for detecting serum specific anti-Ag85B and IFN-γ/IL-2 produced by spleen cells using ELISA. Simultaneously, the levels of CD80 and CD86 on macrophages which were stimulated by Ag85B or TAT-Ag85B protein were measured using flow cytometry. Subsequently, the rest of the mice were intravenously injected with virulent Myobacterium tuberculosis H37Rv and their bacterial loads in the lung and spleen were determined 1, 2, 4 and 8 weeks after infection. Moreover, pulmonary pathological changes were observed by HE staining at 8 weeks after infection.

RESULTS

Ag85B and TAT-Ag85B proteins were obtained successfully. Compared with Ag85B, higher titers of IgG antibodies and the levels of IFN-γ and IL-2 were induced by TAT-Ag85B. Lower bacterial loads in the lung and spleen and smaller scale of pulmonary lesion were found in mice immunized with TAT-Ag85B than those in Ag85B-treated mice. In addition, TAT-Ag85B stimulated higher CD80 and CD86 expressions on macrophages.

CONCLUSION

TAT-Ag85B protein is an efficient vaccine that induces a strong Th1 immune response and provides a good protection against Myobacterium tuberculosis infection. The mechanism of the subunit vaccine may be partially explained as the enhanced capability of antigen-presentation of macrophages.