Construction And Biodistribution Of Bivalent Expressing Avian Influenza DNA Vaccine Plasmid In BALB/c Mice

Avian influenza, caused by avian influenza A virus(AIV), is a virulent infectious disease in poultry. The disease has caused huge economic losses in poultry industry and posed great threat to public health as well. Vaccination is one of the most effective and costbenefit interventions for reducing the mortality and morbidity in domestic birds. DNA vaccine has many advantages over tranditional ones, including: low production costs, significant immune effect, easy to production and preservation. In our previous studies, we have developed successfully an effective DNA vaccine pCAGGoptiHA5 expressing the HA gene of H5 AIV A/Goose/GuangDong/1/96(H5N1) [GS/GD/1/96(H5N1)]. However, the feasibility of avian influenza bivalent or multivalent DNA vaccine and the distribution of antigen proteins in tissues of vaccinated animals were not well documented.To construct avian influenza bivalent DNA vaccines, the enhanced green fluorescent protein(eGFP) gene was separately inserted into DNA Vaccine plasmid pCAGGoptiHA5 as the report gene by using double promoters, the internal Ribosome entry site(IRES) and protein Linker, and then the doublepromoted plasmid pH5-eGFP, IRES mediated plasmid pHIE and fusion expression plasmid pH6 E were successfully constructed and verified by by restriction enzyme digestion and sequencing. The plasmids were then transfected into 293 T cells by cationic liposome method.The expression of HA and eGFP were detected by immunofluorescence assay(IFA) and western blot. The results showed that these three bivalent expressing plasmids express HA and eGFP correctly. The plasmids pH5-eGFP and pHIE expressed the HA and eGFP separately at different sites in cells, while the two proteins were fused together and located on the cell membrane in the plasmid pH6 E.The results indicate that the plasmids pH5-eGFP and pHIE have the potential of being the tools for developing bivalent avian influenza DNA vaccine.To understand the distribution of antigen proteins in tissues of vaccinated animals by the DNA vaccine, we constructed a fusion expression plasmid pH6 L with luciferase gene as the report gene, then the recombinant plasmid were transfected into 293 T cells by cationic liposome method. The expression of recombinant plasmid in 293 T cells was detected using IFA and western blot.The recombinant plasmid pH6 L and the plasmid pLuc that express luciferase were injected to BALB/c mice separately through intramuscular route or electroporation. To visualize the expression of the DNA vaccine under an animal in vivo imaging system, mice were injected with D-luciferin intraperitoneally at XX days post inculation. Tissue samples of mice were prepared for detecting the expression of target proteins by using immunohistochemical(IHC). Serum samples were collected for HI antibody test. The results of IFA and western blot indicated that the recombinant plasmids express the HA protein and luciferase correctly. Visible luminescence was found at the injection site of the immunized mice with electroporation appear at 3 hours post inoculation, while in the immunized mice without electric shock that was not observed untill 1 week post inoculation. The expression of HA was detected in the spleen of immunized mice indicated by the IHC assays. The HI antibodies were detected in electric shocked mice at 1 week post inoculation and in non-electric shocked mice at 3 weeks post inoculation.In conclustion, the study show that the plasmid pH5-eGFP and pHIE have the potential to be the tools for developing bivalent DNA vaccine. Electroporation can improve the immune effects by promoting an earlier expression of antigen proteins. The visualization of the expression of antigen proteins will enhance the understanding of the immue mechanism of DNA vaccine. The efficiency of the bivalent DNA vaccine should be further evaluated in the following studies.