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Objective To explore the effect of MPT64 antigen from Mycobacterium tuberculosis on RAW264. 7 macrophages and the related mechanism. Methods MPT64 was purified after expression in E. crSi and verified by Western blotting analysis. The RAW264. 7 differentiation was induced by phorbol myristate acetate (PMA) and the resultant cells were divided into three groups according to different treatments: negative control, purified protein derivative of BCG(BCG-PPD) and BCG-PPD+MPT64 treatment groups. After 16 h incubation, flow cytometry was used to examine apoptosis of macrophages, and the levels of TNF-α and IL-10 in the supernatants were determined by ELISA. Results BCG-PPD treatment induced apoptosis of RAW264. 7 macrophages, and compared with BCG-PPD group, the apoptotic level of macrophages was significantly lower in BCG-PPD+MPT64 group (P<0. 05). We also found that the supernatant TNF-a level in the BCG-PPD group was significantly higher than that in negative group (P<0. 01) and the IL-10 levels were not significantly defferent between the two groups. Compared with BCG-PPD group, the IL-10 levll was significantly increased in BCG-PPD + MPT64 group (P<0. 01) and the TNF-a levels were not significantly different between the two groups. Conclusion MPT64 may act as a virulence factor and can inhibit the apoptosis of macrophages induced by BCG-PPD, which is probably through increasing IL-10 level.
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Objective: To clone the Ag85 A antigen gene of Mycobacterium tuberculosis and express it in E. coli k802. Methods: The Ag85 A gene was amplified from the genome of Mycobacterium tuberculosis by PCR and was inserted into prokaryotic expressing vector pGEX5T to construct pGEX5T-Ag85A recombinant plasmid. The expression of Ag85A protein in E. coli k802 was induced by IPTG. The expressed protein was purified by affinity chromatography and the antigenicity of the expressed protein was confirmed by Western blotting assay. Results: A band about 0.9 kb in length was obtained by PCR and the recombinant plasmid pGEX5T-Ag85A was successfully constructed. A new band about 58 000 in length was observed after IPTG induction in E. coli. The relative molecular weight of the expressed protein was consistent with that expected. A single protein band of 58 000 in length was obtained after purification. The expressed protein could be specifically recognized by the sera of patients with tuberculosis patients. Conclusion: The Ag85A gene of Mycobacterium tuberculosis has been successfully cloned and expressed in E. coli k802, which paves a way for further studies on diagnosis and therapy of tuberculosis.
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Objective: To modify the mono-antigen DNA vaccine of Mycobacterium tuberculosis with ubiqutin, so as to obtain more potent immune response. Methods: We constructed Mycobacterium tuberculosis MPT64 antigen DNA vaccine (pM) and ubiquitin-MPT64 fusion gene DNA vaccine (pUM). The constructed DNA vaccines were intramuscularly inoculated into female BALB/c mice separately. The serum antibodies(including IgG, IgG1,and IgG2a), cytokines (IFN-γ, IL-4) and cytotoxic T lymphocyte(CTL) response were determined in immunized mice. Results: The IgG level in the pM group was higher than that in the pUM group(P<0.01) and the ratio of IgG2a/IgG1 in the pM group was lower than that in the pUM group([2.16±0.3] vs [4.48±0.4],P<0.05). The IFN-γ level was higher (P<0.01) and the IL-4 level was lower (P<0.01) in the pUM group than those in the pM group. Furthermore, the pUM group had higher CTL activity than the pM group. It was indicated that the fusion gene DNA vaccine induced weaker humoral immune response but stronger cellular immune response compared to single gene DNA vaccine. Conclusion: The fusion gene DNA vaccine constructed in the present study might be more effective for prevention against tuberculosis than the single gene DNA vaccine.