| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 99 === The recent outbreaks of highly pathogenic influenza in Asia and spread of the disease worldwide highlight the need to redefine conventional immunization approaches and establish effective mass vaccination strategies to face global pandemics. For this reason, this study developed the chitosan-based multiple-DNA vaccines mediated by intranasal vaccination to against influenza A virus infection. We used biological compatibility chitosan as vehicle for nasal delivery. The chitosan nanoparticles size was 281.1±3.76 nm and zeta potential was 47.3±1.24 mV. The physical characteristics of chitosan nanoparticles were stable after six-weeks storage. The optimal ratio of the plasmid DNA (pGFP-N1) to chitosan for maximal transfection efficiency, cell viability and DNA binding capacity was molar ratio = 5 in the BHK-21 cells. Then, the formulation of the chitosan-pDNA (p3224/luc) complexes was applied to the nasal of BALB/c mice. After 5 days, we could find the luciferase protein activity in lung. DNA vaccines (pCJ-3/NA, M1 and NP) was constructed and expressed in vivo on respiratory tract epithelial cell of BALB/c mice. Influenza A virus amplified by chicken eggs and the LD50 was 6.8x102 TCID50/mice. After influenza A virus challenge with 5xLD50, mice were dead at day 9 post-infection and the anti-virus immunity toward into cytotoxic T cell (CTL) response. Finally, the prophylactically intra-nasal immunized mice with chitosan-pCJ-3/NA, M1and NP DNA vaccine got the 22%, 12% and 11% survival rate, respectively. Results show that intranasal immunization with chitosan carrier can induce better immune response than without. But the protective effect is still low; the future work can add the CPG motif adjuvant to increase the immune protection of the chitosan-based DNA vaccine mediated by intranasal vaccination.
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