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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30240

Title: 以幾丁聚醣搭載流感病毒DNA疫苗經鼻腔免疫對抗A型流感病毒感染之探討
Study of the chitosan-based DNA vaccines mediated by intranasal vaccination against influenza A virus infection
Authors: Kuang-Yo Chang
鄭光佑
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 流感病毒
Influenza A Virus
Date: 2011
Issue Date: 2011-11-25T07:37:54Z
Abstract: 近年來爆發了高致病性流行性感冒,疫情蔓延到了全世界。這使我們必須重新且全面地檢視傳統的免疫方式,並建立一個新型有效的免疫策,以因應全球性的大流行。因此,本篇論文將發展DNA疫苗對抗流感病毒感染,並利用生物相容性之幾丁聚醣做為鼻腔傳遞系統之載體。在本次的實驗中所製備之幾丁聚醣奈米粒之粒徑為281.1±3.76 nm,而表面電荷為47.3±1.24 mV。此外,幾丁聚醣奈米粒儲存六週後,其粒徑與表面電荷均無明顯改變,顯示其物理特性穩定。接著,使用不同比例之幾丁聚醣-DNA複合體對幼倉鼠腎細胞進行轉染效率、細胞毒性與DNA胞裹效率分析,發現理想之幾丁聚醣-DNA 莫耳數比為5,並將此比例應用於小鼠鼻腔免疫傳輸上。動物實驗顯示,幾丁聚醣與p3224/luc形成複合體經鼻腔免疫5天後,於肺臟偵測到最高之luciferase活性。在基因重組流感DNA疫苗的部分,已建構了pCJ-3/NA, M1 與 NP並證實鼻腔傳遞後DNA疫苗主要表現於肺臟呼吸道上皮細胞。另外,在建立A型流感動物模式的部份,流感病毒利用雞胚蛋進行繁殖並確立其對小鼠之半致死劑量 (LD50) 為6.8x102 TCID50/mice。經5xLD50 攻毒小鼠後,於感染後第九天全數死亡,分析其抗病毒機制主要為細胞毒殺(CTL)反應。最後在免疫保護效果評估上,僅幾丁聚醣包裹DNA疫苗之組別小鼠存活,其保護率依照抗原基因分別為NA、M1與NP分別為22%、12%與11%。結果說明,幾丁聚醣做為經鼻免疫之載體確實能誘發較佳之免疫保護效果,但由於保護效果仍偏低,因此未來可能需要添加佐劑CPG motif,增加免疫保護之效果。
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.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98320006
http://ntour.ntou.edu.tw/handle/987654321/30240
Appears in Collections:[食品科學系] 博碩士論文

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