| ObjectiveTo construct the DNA vaccines encoding MTb antigens including Ag85B, MPT64 and the Ag85B-MPT64 fusion protein (AM), to establish the animal model of tuberculosis in mice, by which to estimate the immunotherapeutic effects of DNA vaccination on the mice tuberculosis. Meanwhile to study the adjuvant effect of psIL-12, which is an eukaryotic plasmid encoding mice Interleukin-12, on the DNA vaccines and to estimate the therapeutic effects of DNA vaccines cooperated with the tuberculosis chemotherapy reagent. Methods1. The eukaryotic expression plasmids pcDNA/Ag85B,pcDNA/MPT64 and pcDNA/AM were constructed. The recombinant plsmids were transfected into COS-7 cells respectively and to test the expressed target proteins by RT-PCR, ELISA and dot blotting. The C57BL/6 mice were immunized with pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/AM respectively, then the total IgG, the proliferation of splenocytes and IFN-γlevel were detected by MTT and ELISA to analyze the specific immune responses induced by DNA vaccination. 2. C57BL/6 mice were infected intravenously (i.v.) via the lateral tail vein with MTb H37Rv strain. Four weeks after infection, the lung and spleen tissues were harvested to have the acid-fast staining, HE staining and MTb culture.3. Four weeks after infection, therapy was initiated by injecting pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/AM in saline into the quadriceps muscle of each hind leg, on four occasions at 2-week intervals. The lung and spleen tissues were harvested to quantitate the viable mycobacteria in the organs, to estimate the organ MI and have the histopathological examination. The level of IFN-γ, TNF-αand IL-4 in splenocytes were investigated by ELISA kit.4. Four weeks after infection, therapy was initiated by injecting DNA plasmids and psIL-12 mixture (50:50) in saline into the quadriceps muscle of each hind leg, on four occasions at 2-week intervals. The lung and spleen tissues were harvested to quantitate the viable mycobacteria in the organs, to estimate the organ MI and have the histopathological examination. The level of IFN-γ, TNF-αand IL-4 in splenocytes were investigated by ELISA kit.5. Four weeks after infection, the INH,PZA cooperated with pcDNA/Ag85B DNA vaccine were used against mice tuberculosis. The lung and spleen tissues were harvested to quantitate the viable mycobacteria in the organs, to estimate the organ MI and to observe the tissue histopathological changes.Results1. pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/AM were constructed and the inserted target genes were confirmed by restriction enzyme analysis as well as DNA sequencing. The supernatant of COS-7 cell cultures transfected with pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/AM showed positive reaction to rAg85B and rMPT64 antibodies by ELISA. The antibody titer against PPD, the proliferation of antigen-specific lymphocyte and IFN-γproduction in DNA vaccination group were higher than those of the control groups(p<0.05).2. Four weeks after infection, MTb was cultured from the lung and spleen tissues of the infected mice. MTb was observed in those tissues with acid-fast staining. The histopathological examination showed that the alveoli and interalveolar septae disappeared along with the massive infiltration of macrophages, most of which had abundant foamy cytoplasm.3. The mice injected with pcDNA/Ag85B had significantly reduced number of CFU and MI of their lungs and spleens compared to those of the control injected with empty plasmid or saline (P<0.01, P<0.05). The pulmonary lesions of pcDNA/Ag85B group were slight and there was an aggregation of epithelioid macrophages. The production of IFN-γand TNF-αof pcDNA/Ag85B group were significantly higher than those of the control groups(P<0.05, P<0.01). 4. The psIL-12 group had significantly reduced number of CFU and MI compared to those of the control groups (P<0.05, P<0.05). The pulmonary lesions of psIL-12 group were slight and limited. The production of IFN-γand TNF-αof psIL-12 group we...
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