| ObjectiveThe study aimed to construct the fusion gene DNA vaccine encoding Ag85B and MPT64 linked with (Gly4Ser)3 linker, amplified from M.tuberculosis H37Rv chromosomal DNA, and, to explore the immune responses and protective efficacy of the DNA vaccine in mice infected with MTb. It would lay a foundation for a new tuberculosis vaccine and provide data for immunoprophylaxis and immunotherapy. Methods1. Construction of eukaryotic expression plasmids The genes encoding Ag85B(ag85b), MPT64(mpt64) and AM[Ag85B and MPT64 fusion gene linked with (Gly4Ser)3 linker] were obtained from M.tuberculosis H37Rv chromosomal DNA by PCRamplification,with primers designed to generate HindIII and BamHI restriction sites at the 5'and 3'ends of the amplified fragments respectively. The genes were cloned into unique HindIII and BamHI sites of the pcDNA3.1(+).The recombinant plasmids were identified with DNA sequence and endonucleases.2. Expression and purification of Ag85B and MPT64 recombinant proteins in E.coli BL21, and fabrication of anti-Ag85B and anti- MPT64 antibodies.The ag85b and mpt64 gene fragements were isolated from the pcDNA/Ag85B and pcDNA/MPT64 by restriction endonucleases, and subcloned into prokaryotic expression vector pET32a, then recombinant plasmids pET/Ag85B and pET/MPT64 were constructed. The recombinant plasmids were expressed in E.cli BL21, and its expressive products were purified. BABL/c mice were immunizated with the purified proteins.3. Expression of the DNA vaccines in COS-7 cell lines. COS-7 cell lines were transfected with plasmid vectors using cationic liposome respectively. 48 hours later, the fusion protein, Ag85B and MPT64 proteins expressed in COS-7 cell lines were determined by RT-PCR, ELISA and dot blotting. 4. Immunogenicity of the DNA vaccines. C57BL/6 mice were intramuscularly immunized with the saline,plasmid vector, pcDNA/Ag85B, pcDNA/MPT64, pcDNA/AM and pcDNA/BM respectively. The specific antibody levels were determined by ELISA, and spleen lymphocyte proliferation and IFN-γ levels in response to antigen restimulation was measured by MTT and ELISA in vaccinated mice respectively. 5. The evaluation of the protective effects of DNA vaccines against M. tuberculosis. C57BL/6 mice were intramuscularly immunized with the DNA vaccines or BCG (i.d.). The mice were challenged with 106CFU H37Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed respectively, and the number of CFU in organs and histopathologic changes was determined. The antibody level, IFN-γ,IL-4 and the survival time in all of the mice were evaluated .Results1.Recombinant pcDNA/Ag85B, pcDNA/MPT64, pcDNA/AM, pcDNA/BM were constructed and the inserted target genes were confirmed by restriction enzyme analysis and DNA sequencing. The fusion gene was sequenced, the mutation rate was 0.11%(2/1707) and mutation was nonsense.2.The pET/Ag85B and pET/MPT64 were expressed in E.coli BL21, and its expressive products were analyzed by SDS-PAGE and identified by Western-blot. Antibody titer of anti-Ag85B and anti-MPT64 was 1:64 and 1:32 respectively.3.The supernatant of COS-7 cell cultures transfected with pcDNA/AM showed positive reaction to both Ag85B antibody and MPT64 antibody by ELISA. 4.The antibody titer against PPD, antigen-specific lymphocyte proliferation and IFN-γproduction in Ag85B, MPT64 and AM DNA vaccine groups were higher than that of other groups (p<0.05).5.The sera from immunized C57BL/6 mice were examined for PPD antibody by ELISA at 6 weeks after challenge with M.tuberculosis H37Rv. Antibody titer of pcDNA/Ag85B+pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (p<0.05). Spleen lymphocytes from immunized mice were restimulated in vitro with PPD.The level of IFN-γproduced by spleen lymphoc...
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