Experimental Study On DNA Vaccine Expressing Hemagglutinin And Nucleoprotein Gene Of H5N1 Influenza Virus From Tigers

Objective: Eukaryotic expressing plasmids were constructed expressing the hemagglutinin (HA) and neucleoprotein (NP) gene of H5N1 influenza virus. The eukaryotic expressing plasmids were used to immunize the mouse, the humoral immunity and cell-mediated immunity response of the immunized mice were detected and the protective rates of the eukaryotic expressing vector was analyzed. The work done here provide a valuable theoretical and technical bases for the nucleic acid vaccine development against mammalian H5N1 subtype influenza virus.Method: Ordinary molecular methods were used to construct eukaryotic expressing vectors, pVAX-HA and pVAX-NP expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger. Dual-expressing vector pIRES2-IL-18-HA expressing HA gene of H5N1 subtype influenza virus and IL-18 gene of feline was also constructed. The protein expressed by eukaryotic vectors in BHK-21 cell was detected by indirect immunofluorescence assay and indirect ELASA. Mice were inoculated with four eukaryotic expressing vector intramuscularly for 4 times with a interval of 15 days, 7 immune groups were used as control: immunized with pVAX-HA, pVAX-NP, pVAX-Ha and pVAX-NP, pIRES2-IL-18-HA, IRES2-IL-18-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP, PBS, respectively. 15 days post inoculation, the antibody titers of the mice in each group against AIV were detected by indirect ELASA, MTT method was used to detect the cell-mediated immunity response. The mice in each group were inoculated with highly pathogenic H5N1 subtype influenza virus, in order to measure the protective rates.Conclusion: The HA and NP genes of H5N1 subtype influenza virus were chosen as the target genes to be expressed by eukaryotic expressing plasmids. The proteins expressed all represented biological activity and had the ability to induce the immune protective effects of mice. Inoculated with high titer of highly pathogenic virus (10000 MLD₅₀/each), the mice vaccinized with pVAX-HA produced the highest protective rate, which is 60%. This research provide a valuable theoretical and technical bases for the nucleic acid vaccine development against mammalian H5N1 subtype influenza virus.Result: Four eukaryotic expressing plasmids, pVAX-HA, pVAX-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP, expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger were constructed sucessfully. These four eukaryotic expressing vectors all have the ability to express HA and NP protein with biological activity in the mammalian cell, BHK-21. Mice in each immunized group were inoculated high titer of highly pathogenic virus (10000 MLD₅₀/each), the protective rates of pVAX-HA, pVAX-NP, pVAX-HA and pVAX-NP, pIRES2-IL-18-HA, pIRES2-IL-18-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP, PBS immunized group were 60%, 20%, 60%, 20%, 0%, 20%, 0%, repectively.