Enhancement Of A Plasmid DNA Vaccine By Incorporating Immunostimulatory Motifs

Hsp65 is known to be a good antigen with protective and therapeutic activities against tuberculosis when expressed from plasmid DNA vaccines. However, improved efficacy is desirable. To construct the recombinant prokaryotic expression vector containing Heat shock protein 65 (Hsp65) coding gene of Mycobacterium leprae, and to express and purify the fusion protein and prepare Anti-Hsp65 mouse polyclonal antibody; To improve the humoral and cellular immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen; To involve of intron A into eukaryotic expression vector to improve antigen expression and induce enhanced immunogenicity of DNA vaccine in mice. Immunogenicity was evaluated by ELISA,ELISPOT and FACS.The results are summarized as follows:1,The M. leprae hsp65 gene was amplify and its recombinant prokaryotic expression vector was constructed successfully. The recombinant protein was induced and purified with relative molecular mass 65 kDa, the titer of polyclonal antiserum was found to be 1:12,800 by ELISA and was verified to have a good immunogenicity by Western-blotting;2,When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for IFN-α,IL-12,IL-17A and IL-6 and smaller increases for IL-4 and CD86. Immunized mice showed substantially increased serum Anti-Hsp65 antibody levels and IFN-y production by spleen cells;3,Compared with non-intron pVAX1hsp65, the recombinant plasmid pCMV4.0hsp65 with intron A caused higher expression level of Hsp65 in 293T cells, and induced enhanced Thl type immune response, which was defined as higher level of Anti-Hsp65 specific Ig2a and more IFN-y-secreting CD4+ and CD8+ T lymphocytes.Under these verification, the recombinant M. leprae Hsp65 protein was expressed and purified successfully in vitro, and the Anti-Hsp65 mouse polyclonal antibody with high titer and good immunogenicity was prepared as well; DNA vaccines may be made more highly immunogenic by including transcribed stimulatory sequences; Intron A can improve antigen expression and induce enhanced immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.