| In this experiment, to construct the PPV and JEV DNA vaccine, and then to screen out the highly effective one for its immunogenicity,PPV VP2 and JEV E10 were connected, which were cloned into pcDNA-3.1. And the recombinant plasmid pcDNA.VE was obtained. The poIFN-y gene was amplified by a pairs of specific primers and inserted into plasmid pcDNA.VE. The plasmid pcDNA.VEI was constructed and coexpressed. Transfection to the vero cells was mediated by liposome, for its expression was observed by indirect immuno-fluorescence assay. The Balb/c mice were immunized with this DNA vaccine named pcDNA.VEI, pcDNA.VE within no IFN-y expressed, Inactivated PPV Vaccine, JE live Attenuated Vaccine, and pcDNA3.1 as the blank control groups. The mice were immunized 2 times and immune indexes were tested per 2 weeks. The stimulation of spleen lymphocytes, the level of two sorts of antibodies introduced by pcDNA.VEI and the number of T Lymphocyte Subsetsweres were measured.The result showed that the DNA vaccine was successfully constructed and the vero cells transfected by it were observed by fluorescence microscope. The stimulation of spleen lymphocytes was proved on the DNA vaccine.The ELISA results showed that the level of two sorts of antibodies introduced by pcDNA.VEI was higher than the IFNy-negative control group, which was not obvious in the first 2 weeks, but the level steadily increased and was much higher at the 4th week. The T Lymphocyte Subsets were steadily induced by group of pcDNA.VEI, and the rates of CD4+/CD8+ induced by which were all higher than group of pcDNA.VE after 2 weeks to the first immunizing. The results showed that the DNA vaccine was successfully constructed and enhanced the level of specific humoral and cellular inmmne response elicited in mice, and the safty could be proved.As the promising immune adjuvant gene, IFNy cloned in this DNA vaccine had the effect of immunologic enhancement. |
