中国生物工程杂志

Objective Construct eukaryotic expression plasmid pVAXⅠ- P1-2A containing capsid protein precursor P1-2A gene of Asia-ⅠFMDV ,and evaluate cellular and humoral immunity level after immunization mice with pVAXⅠ- P1-2A. Methods We amplied the coding region of FMDV P1-2A by RT-PCR and inserted it to Vector pMD18-T. Eukaryotic expression plasmid pVAXⅠ- P1-2A was constructed by connecting pMD18-T-P1-2A and pVAXⅠdigested by double restriction endonuclease EcoRⅤand XbaⅠ. We transfected the right recombinant plasmid confirmed by endonuclease digestion into the Hela cell ,and perform IFA to assure the right expresstion. After this, mice serological specific antibody test ,T lymphocyte proliferation assay and IFN-γ ELISPOT test were carried out. Results The result of restriction endonuclease digestion was accordance with the anticipated objective strap size . IFA showed that there are flavovirens fluorescence in cells transfected with pVAXⅠ-P1-2A ,and confirmed the expression of P1-2A gene in the HeLa cell; The result of mice immunization test indicated that there are stronger cellular and humoral immunity response , and more T lymphocyte and the cells that produce IFN-γin immunized mice than the control groups (P<0.05). The indirect ELISA test manifested that the antibody level was higher remarkably than the control groups (P<0.05) in the 14th day after immunity. Conclusion we have successfully constructed eukaryotic expression plasmid pVAXⅠ- P1-2A ,and confirmed that specific humoral immunity and cellullar immunologic response can be induced in the group injected with recombinant plasmids with mice immunization test.