臺灣博碩士論文加值系統

套膜E蛋白是日本腦炎病毒粒子表面最主要的抗原蛋白，具有引發中和性抗體的能力。因此在研發新的日本腦炎疫苗中，常是以套膜E蛋白為主要的設計方向。套膜E蛋白的結構可分為三個區域蛋白，其中第三區域蛋白(D3)能獨立折疊成近似免疫球蛋白之立體構形，並含有多個中和抗原決定位。本論文的研究目之一，在利用大腸桿菌大量表現日本腦炎之第三區域蛋白(D3)，探討其抗原免疫性。另一方面，利用昆蟲細胞桿狀病毒表現系統表現日本腦炎全長套膜E蛋白，並比較套膜E蛋白在大腸桿菌和昆蟲細胞桿狀病毒表現系統的差異性。由實驗結果得知，第三區域重組蛋白(D3)以FCA/FIA(freund’s complete adjuvant / freund’s incomplete adjuvant)乳化施打免疫ICR雜系小鼠後， 在免疫血清1:5的稀釋下，免疫血清對日本腦炎病毒產生高達75%的中和效果(PRNT50= 40)。利用昆蟲細胞桿狀病毒表現之全長套膜E蛋白，施打免疫ICR雜系小鼠後，在免疫血清1:5的稀釋下，免疫血清對日本腦炎病毒產生高達84%的中和效果(PRNT50= 80)。最後本實驗以微脂粒當作佐劑，包覆第三區域重組蛋白(D3)。經注射免疫實驗動物後，發現帶正電微脂粒包覆後的重組蛋白，對JEV病毒有高達60%的中和效果(PRNT50= 10)。由攻毒試驗的結果得知，以帶正電微脂粒包覆的存活率較以FCA/FIA乳化及不帶電與帶負電微脂粒包覆的存活率為佳。綜合以上的實驗結果得知，本篇論文提供了一個未來製作日本腦炎重組蛋白疫苗與新型佐劑配方的可行性。

The envelope (E) protein is the major antigen of Japanese encephalitis virus (JEV) capable of inducing neutralizing antibodies. Thus, JEV E protein has been mainly chosen in the design of novel JEV vaccines. E protein contains three distinct domains (domains I, II, and III). The domain III (D3) contains several neutralizing epitopes and can be independently folded as a unique Ig-like conformational structure. One of the major goals in this thesis is to investigate the immunogenicity of the recombinant D3 protein produced in Escherichia coli. In addition, a full-length E protein of JEV was cloned into baculovirus vector and expressed functionally in cultured insect cells. The immunogenicity of the recombinant D3 protein coupled with FCA/FIA (freund’s complete adjuvant/freund’s incomplete adjuvant) immunized in ICR mice showed a 75% neutralizing titer at 1: 5 diluted sera (PRNT50 = 40). Immunization of the full-length recombinant E protein expressed in baculovirus/insect cell system coupled with FCA/FIA showed a 84% neutralizing titer at 1:5 diluted sera (PRNT50 = 80). Various formulations of liposome adjuvant coupled with the D3 recombinant protein were investigated for immunization studies. The cationic liposome adjuvant induced a 60% neutralizing titer at 1: diluted sera (PRNT50 = 10). After challenges, the survival rates for ICR mice immunized with the D3 recombinant protein coupled with cationic liposome were higher than that coupled with FCA/FIA and with anionic and neutral liposomes. These results can provide important information for further development of JEV recombinant protein vaccines and/or new adjuvant formations.

第一章緒論
1.1日本腦炎………………………………………………1
1.2日本腦炎病毒…………………………………………3
1.3日本腦炎病毒套膜E蛋白……………………………5
1.4新型日本腦炎病毒的研發……………………………7
1.5佐劑在疫苗的發展……………………………………9
1.6微脂粒佐劑的發展……………………………………11
第二章材料與方法
2.1病毒與菌株
2.1.1日本腦炎病毒…………………………………14
2.1.2大腸桿菌菌株…………………………………15
2.1.3昆蟲細胞桿狀病毒表現系統……………………16
2.2細胞株……………………………………………16
2.3日本腦炎病毒E基因相關重組蛋白抗原性鑑定與表現
情形………………………………………………17
2.4重組蛋白製備與純化
2.4.1重組蛋白製備………………………………19
2.4.2重組蛋白純化…………………………………20
2.5蛋白質濃度測定………………………………………21
2.6SDS-PAGE 電泳………………………………………22
2.7西方墨點法(Western Blotting)……………………23
2.8動物免疫實驗…………………………………………24
2.9血清中和試驗…………………………………………25
2.10微脂粒製備……………………………………………27
2.11包覆效率及抗原性鑑定………………………………27
第三章結果
3.1日本腦炎病毒套膜E基因相關重組蛋白的表現…….29
3.2日本腦炎病毒套膜E基因相關重組蛋白製備、純化與
抗原性鑑定…………………………………29
3.3日本腦炎病毒套膜E基因相關重組蛋白的免疫性…31
3.4D3融合重組蛋白的劑量與免疫動力曲線……………32
3.5微脂粒疫苗佐劑的應用………………………………33
3.6微脂粒包覆的免疫性…………………………………34
3.7免疫實驗動物保護試驗………………………………35
第四章討論
4.1日本腦炎病毒套膜E基因相關重組蛋白的表現….36
4.2日本腦炎病毒套膜E基因相關重組蛋白的免疫性…38
4.3微脂粒佐劑…………………………………………39
第五章 結論……………………………………………………41
參考文獻…………………………………………………………….42
圖表…………………………………………………………………50

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