人類為D型肝炎病毒(HDV)之主要感染宿主，感染後往往會導致猛爆性肝炎或慢性肝病等等，但由於目前缺乏有效的方法來生產HDV蛋白質，故尚未有有效的疫苗或診斷試劑問世。因此為了解決此問題，我們期望建立一套桿狀病毒/哺乳動物細胞之表現平台，以生產具有正確轉譯後修飾之HDV蛋白質。在本研究中，我們首先著眼於以D型肝炎大型抗原(L-HDAg)作為疫苗生產之探討。實驗發現表現his-tagged L-HDAg (L-HDAgH)之重組桿狀病毒能有效的轉導哺乳動物細胞BHK，其轉導效率能達到90%以上，且其表現量明顯高於以質體轉染所得，在添加10 mM丁酸納下可得到約19 □g/106 cells。除此之外，L-HDAgH不但能正確的分佈於細胞核中，且具有正確的異戊二烯化修飾，並能在denature的狀況下藉由IMAC親和力層析達到部份純化的效果。另一方面，我們更進ㄧ步嘗試以桿狀病毒/哺乳動物細胞表現系統生產及分析D型肝炎類病毒顆粒(HDV VLP)。在選擇適當的病毒來生產HDV VLP後，我們發現病毒的共同轉導比例會造成L-HDAg及HBsAg相對表現量的差異，進而影響HDV VLP的性質。較高的Bac-GD對Bac-GS2之MOT比例會使細胞內L-HDAg對HBsAg的相對量提高，導致HDV VLP內有較多之L-HDAg；因此進ㄧ步使組成之HDV VLP有較大的粒徑分佈、較大的密度、以及較高的L-HDAg對HBsAg之重量比。總括來說，本研究證明了桿狀病毒/哺乳動物細胞表現系統確實可提供作為蛋白質或VLP之生產平台，並能有效的應用於D型肝炎的研究上。

Human is the host of Hepatitis delta virus (HDV), which can cause severe acute liver inflammation or chronic liver diseases. Currently there is no effective vaccine or diagnostic reagent for HDV due to the lack of a proper method to efficiently produce HDV proteins. To this end, the baculovirus/ mammalian cell system was explored as a useful tool to produce HDV proteins with proper post-translational modification. In this study, we first focused on the production of large hepatitis delta antigen (L-HDAg) to be a promising vaccine candidate against HDV. The recombinant baculovirus which expresses his-tagged L-HDAg(L-HDAgH) transduced the BHK cells with efficiencies higher than 90%. The expression level was significantly higher than that could be obtained by plasmid transfection and was further enhanced 3-fold to □19 □g/106 cells by the addition of 10 mM sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated. Moreover, L-HDAgH can be partially purified by denaturing Ni2+ chelate affinity chromatography. In addition, we also employed the baculovirus/ mammalian cell system to produce and characterize hepatitis delta virus-like particles (HDV VLP). After selecting the suitable virus for VLP production, we found that the relative baculovirus dosage, and hence the relative expression levels of L-HDAg and HBsAg, profoundly influenced the properties of HDV VLP. The higher MOT ratio of GD/GS2 increased the relative amounts of L-HDAg to HBsAg within the cells, thus leading to the incorporation of more L-HDAg into the HDV VLP. This, in turn, caused larger VLP size, higher density and larger weight ratio of L-HDAg/HBsAg in the particles. In summary, this study demonstrated that the baculovirus/ mammalian system provides a suitable way to produce proteins and VLP and apply on HDV research.

目錄 I
圖目錄 IV
表目錄 V
第一章 緒論 1
1.1 昆蟲桿狀病毒與昆蟲桿狀病毒表現載體系統 1
1.2 桿狀病毒/哺乳動物細胞表現系統 2
1.3 D型肝炎病毒(HDV) 4
1.4 類病毒粒子(VLP) 6
1.5 研究動機 7
第二章 實驗材料與方法 10
2.1 細胞培養 10
2.1.1 哺乳動物細胞與培養基 10
2.1.2 昆蟲細胞與培養基 10
2.2 重組桿狀病毒 10
2.2.1 重組桿狀病毒載體建構 10
2.2.2 重組桿狀病毒放大 11
2.2.3 終點稀釋法定量病毒濃度 12
2.3 桿狀病毒/哺乳動物細胞表現系統 12
2.3.1 桿狀病毒轉導哺乳動物細胞 12
2.3.2 二倍稀釋法定量病毒濃度 13
2.4 流式細胞儀 13
2.5 蛋白質分析 14
2.5.1 SDS-PAGE 14
2.5.2 西方點墨法(Western blot) 14
2.6 免疫螢光分析 15
2.7 親和力層析法純化L-HDAgH 16
2.7.1 L-HDAgH萃取 16
2.7.2 L-HDAgH純化 16
2.8 Protein assay 17
2.9 初步濃縮HDV VLP 18
2.9.1 超過濾系統(Ultrafiltration) 18
2.10 超高速離心法純化HDV VLP 18
2.10.1 蔗糖緩衝(sucrose cushion)超高速離心 18
2.10.2 氯化銫等密度離心 18
2.11 穿透式電子顯微鏡(TEM) 19
2.11.1 樣品負染 19
2.11.2 免疫金標記 19
第三章 結果與討論(Ⅰ)—D型肝炎大型抗原探討 22
3.1 重組蛋白質表現確定 22
3.2 轉導效率與時間序列分析 23
3.3 重組蛋白質表現量提升 24
3.4 重組蛋白質性質分析 25
3.5 重組蛋白質純化 26
3.5.1 Binding buffer選擇 26
3.5.2 L-HDAgH純化 29
第四章 結果與討論(Ⅱ)—D型肝炎類病毒顆粒探討 38
4.1 病毒選擇 38
4.2 病毒劑量比例探討 41
4.2.1 HDV VLP密度分析 42
4.2.2 HDV VLP粒徑分佈探討 43
4.2.3 HDV VLP粒徑大小確定 44
4.2.4 HDV VLP組成分析 46
4.2.5 HDV VLP探討 48
第五章 結論 59
第六章 未來與展望 61
6.1 L-HDAgH純化最適化 61
6.2 HDV VLP特性分析 61
6.2.1 粒徑分佈與結構探討 62
6.2.2脂質組成分析 62
6.2.3 分解與組裝能力探討 62
6.3應用研究 63
6.3.1 免疫性質探討 63
6.3.2 包覆性質評估 63
參考文獻 64

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