臺灣博碩士論文加值系統

禽流感(Avian influenza)對全世界家禽產業已造成難以估計的經濟損失，而其高致死率也使得發展新疫苗成為控制疫情的主要方式。本實驗的研究目標是以家禽應用為主要導向，利用基因重組的技術，以水生植物浮萍(Lemna minor)表現系統研發安全有效的次單元疫苗，來對抗新型的流感病毒。本研究首先成功地建構出浮萍基因表現及基因轉殖系統，同時也成功地利用基因合成技術將H5流感病毒表面抗原中血球凝集素(HA; hemagglutinin)基因以Lemna gibba (US patent 8022270 B2)最適基因碼進行編碼，並透過農桿菌之轉染分析，經由免疫分析檢測證實利用浮萍植物可正常地表現出血球凝集素蛋白質，且表現的HA蛋白仍具有鷄紅血球凝集活性(HI)且可被PNGase F切割，同時可被構形型之單株抗體辨認，此外，本研究也成功地建立了浮萍疫苗之純化製程，亦即疫苗純化可先透過珠磨法進行破壁，再利用Biologic low pressure chromatography system純化設備即可達到目標疫苗的目的。為提升浮萍株生產效率，本研究於相同體積之培養箱分別置入不同新鮮重量之浮萍進行試驗，據結果顯示加入植種量為10 g m-2時，可得到最高生長速率；為進一步確認血清檢體之抗體量，亦利用血球凝集素之延伸性進行測試，實驗結果發現HA具有專一性，可將流感病毒鑑別出來。另外，在動物試驗方面，本實驗則以表現HA蛋白之浮萍直接製備成疫苗供鷄隻食用，其服用二週時間即可引起高力價(log2≥6.8) 之血球凝集抑制(HI)抗體反應。因抗體力價大於Log2=3之鷄隻即可得完全的保護效果，此結果說明利用水生植物浮萍(Lemna minor)表現生產之重組HA抗原可發展成安全且有效之次單位疫苗供鷄隻使用。
透過此研究成功地發展出安全有效、成本低且適用於水禽場的新一代水生植物浮萍禽流感疫苗，亦可利用水生浮萍作為包覆性疫苗，供於未來疫苗之生產。

Avian influenza virus has posed a worldwide threat to human health, yet there is still no effective vaccine available. This research is to develop a safety and effective recombinant subunit vaccine against pandemic influenza virus by using aquatic plant Lemna. The selected targets is the hemagglutinin i.e.HA. To achieve the highest possible levels of productivity, the target gene codon was altered by gene optimization technology. To further evaluate its potential as a commercial vaccine, in this research we developed a bioprocess for the large-scale production and purification of Lemna-made vaccine with a BioLogic LP System. It’s immunoreactivity was analyzed by Western blotting, ELISA and blocking ELISA using MAb and positive serum. In our results, hemagglutination inhibition tests indicated that the conformation of the aquatic plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. This new research will offer an attractive system for Avian influenza oral vaccines.

明志科技大學碩士學位論文指導教授推薦書………………………………..i
明志科技大學碩士學位論文口試委員會審定書............…………………….ii
明志科技大學學位論文授權書………………………………………………iii
誌謝……………………………………………………………………………iv
中文摘要……………………………………………………………………….v
ABSTRACT...………………………………………………………………...vii
目錄………………………………………………………………………......viii
圖目錄……………………………………………………………………....….x
表目錄………………………………………………………………………....xi
第一章 緒論…………………………………………………………………...1
第二章 文獻回顧…………………………………………………………..….2
2-1疫苗…………………………………………………………………...2
2-1.1口服疫苗…………………………………………………….…2
2-2家禽流行性感冒病毒…………………………………………………4
2-2.1歷史背景……………………………………………………….4
2-2.2宿主範圍……………………………………………………….5
2-2.3病毒特徵……………………………………………………….6
2-2.4病毒結構與功能……………………………………………….7
2-2.4.1聚合酶蛋白（polymerase protein）……………………….7
2-2.4.2血球凝集素（hemagglutinin, HA）………………………8
2-2.4.3核蛋白（uncleoprotein, NP）………………..…………8
2-2.4.4神經胺酸酶（neuraminidase, NA）………………………9
2-2.4.5基質蛋白（matrix proteins, M）……………………….9
2-2.4.6非結構蛋白（nonstructural proteins, NS）………………9
2-3蛋白質表現系統…………………………………………………….12
2-3.1植物表現系統…………………………………………………13
2-3.2以植物表現系統生產蛋白……………………………………14
第三章 實驗方法…………………………………………………………….17
3-1 Lemna浮萍表現系統建立……………………………………….….17
3-2浮萍基因轉殖系統…………………………………………………..17
3-3抗原基因之選擇與基因全合成………………………………….….18
3-4重組抗原之純化製程……………………………...…………….….18
3-5浮萍表達抗原之檢驗分析……………………………...……….….18
3-6動物免疫分析……………………………...…………….…………..19
3-7免疫分析(immunoassay) ……………………………...…………….19
3-8血球凝集及血球凝集抑制試驗………………...…………….……..20
第四章 結果與討論……………………………...…………….…………….21
4-1 Lemna浮萍表現系統建立……………...…………….…………….21
4-2 HA基因全合成之小葉浮萍表現載體建構與蛋白質表現分析…...22
4-3基因改質HA生產浮萍株biomass生產效率……………...……….23
4-4血球凝集抑制試驗……………...…………….……………………..24
第五章 結論……………...…………….…………………………………….26
第六章 參考文獻……………...…………….……………………………….28

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