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dc.contributorWei-Li Hsuen_US
dc.contributorPei-Wen Hsiaoen_US
dc.contributorWen-Jen Yangen_US
dc.contributor.advisorMin-Ying Wangen_US
dc.contributor.authorTseng, Yuan-Pinen_US
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dc.description.abstract流感似病毒顆粒為近年來新一代的流感疫苗,具有類似真實病毒顆粒的外形及抗原性、不具病毒核酸等特點。本研究使用桿狀病毒表現系統在Hi-5昆蟲細胞株表現H1N1亞型A/PR/8/34流感病毒株的血球凝集素蛋白(HA),經由血球吸附試驗證實感染的細胞可表現具有功能的HA膜蛋白,並且胞外的培養基具有血球凝集現象,顯示部分的HA分子被釋放到胞外區域。以病毒感染複數(MOI)最適化試驗,得到細胞以MOI = 0.01感染72小時具有最佳的胞外HA產量,約為64 HAU /50μl (1.2 μg/ml)。在20~60% 蔗糖梯度超高速離心及以動態光散射分析結果證實被釋放到胞外HA分子為顆粒型態,其浮力密度介於1.17~1.20 g/cm3,平均粒徑為174 ± 7 nm。進一步以穿透式電子顯微鏡及免疫金標定證實所觀察到的似病毒顆粒確實含有HA。另一方面,以介質輔助雷射脫附游離-飛行時間式質譜儀 (MALDI/TOF) 分析HA似病毒顆粒分層的組成,發現除了HA蛋白外,還有來自細胞的HSP70、beta-tubulin和cytoplasmic actin以及來自桿狀病毒的gp64蛋白,這些蛋白有可能參與HA似病毒顆粒的釋出過程。動物免疫實驗證實BALB/c小鼠在免疫兩次含0.2 μg或2 μg HA的似病毒顆粒後,皆可產生高力價的血球凝集抑制(HI)抗體 ( >150倍)。綜合上述結果顯示利用桿狀病毒表現系統在Hi-5昆蟲細胞株表現H1N1亞型A/PR/8/34流感病毒株的血球凝集素蛋白可以形成具有免疫原性的HA似病毒顆粒且具有開發成為流感似病毒顆粒疫苗的潛力。zh_TW
dc.description.abstractInfluenza virus-like particles (VLPs) are prominent influenza vaccine candidates because of their similar morphologies and immunogenicities to authentic virions and lack of viral genetic materials. In this study, hemagglutinin (HA) of influenza A/PR/8/34 (H1N1) virus was expressed as a membrane protein, which conferred the infected Hi-5 cells with the function to agglutinate erythrocytes. Strikingly, the hemagglutination was also detectable in the medium, which suggested some HA molecules were released into extracellular environment. Multiplicity of infection (MOI) optimization showed that cells were infected with 0.01 MOI at 72 hours post infection has the highest titer of extracellular HA for 64 HAU/ 50μl (1.2 μg/ml). 20-60% sucrose density gradient ultracentrifugation and dynamic laser scattering analysis were conducted to demonstrate that these extracellular HA molecules were released in a particle form with the buoyant densities between 1.17-1.20 g/cm3 and average diameter of 174 ± 7 nm. The existence of VLPs formed by HA (HA-VLPs) was further confirmed by electron microscopy and immunogold labeling. On the other hand, cell-derived HSP 70, cytoplasmic actin and beta-tubulin and baculovirus gp64 protein were MALDI /TOF-identified in the same fraction of HA-VLPs, which suggested that these proteins might involve in the budding process of HA-VLPs. Animal immunization demonstrated that two doses of VLPs containing 0.2 μg or 2 μg HA can elicit high titers of hemagglutination inhibition (HI) antibody (>150) in all vaccinated BALB/c mice. Taken together, these results demonstrates that using recombinant baculovirus expression system to express influenza virus A/PR/8/34 (H1N1) HA in Hi-5 insect cells is capable to produce immunogenic HA-VLPs and indicate that HA-VLPs represent another promise influenza VLP vaccine candidate.en_US
dc.description.tableofcontents中文摘要 i Abstract ii 目次 iv 表目次 vii 圖目次 vii 第一章 背景介紹 1 第一節 流行性感冒病毒簡介 1 第二節 A型流感病毒 1 第三節 A型流感病毒的血球凝集素 2 第四節 目前人用流感疫苗 2 第五節 流感似病毒顆粒疫苗 3 第六節 流感似病毒顆粒的組裝研究現況 3 第七節 研究動機與目的 4 第二章 材料與方法 5 (一) 限制酶及其他酵素 5 (二) 重組質體pFB-H1、pFB-eHA1h、pET21b-eHA1h∆S構築 5 (三) 勝任細胞(Competent cells)的製備 5 (四) 轉型作用(Transformation) 6 (五) 轉型菌株的篩選與鑑定 6 (六) 專一性 H1亞型 HA抗體 (anti-H1)的製備 6 (七) 點漬法(Dot blot) 6 (八) SDS-聚丙醯胺膠體電泳分析(SDS-PAGE) 7 (九) 西方墨點轉漬法 (Western blot) 7 (十) 固定化金屬親和性色層分析法 7 (十一) 昆蟲細胞來源及繼代 8 (十二) 重組桿狀病毒 Bac-H1 8 (十三) 桿狀病毒 DNA 純化 9 (十四) 桿狀病毒力價測定-病毒斑試驗 ( Plaque assay) 9 (十五) 紅血球吸附試驗(Hemadsorption assay) 9 (十六) 紅血球凝集試驗 ( Hemagglutination test,HA test) 9 (十七) HA蛋白產量最適化 10 (十八) 以磁攪拌培養瓶(spinner-flask)表現HA蛋白 10 (十九) 蔗糖梯度離心–流感VLPs的純化 10 (二十) 定量各分層的HA 11 (二十一) 各分層的總蛋白量 11 (二十二) gp64的分布 11 (二十三) 不同分層感染細胞分析桿狀病毒分布 11 (二十四) 各分層的DNA含量 11 (二十五) 以Quantitative PCR (qPCR) 檢測桿狀病毒力價 11 (二十六) 病毒斑試驗檢測各分層中的桿狀病毒力價 11 (二十七) 動態光散射分析 (Dynamic Light Scattering,DLS) 12 (二十八) 以穿透式電子顯微鏡觀察H1-VLPs 12 (二十九) 專一性 H1 亞型 HA 抗體 (anti-H1) 的純化 12 (三十) 免疫金標定H1-VLP 13 (三十一) 以二次純化方式濃縮H1-VLPs 13 (三十二) H1-VLPs組成分析 13 (三十三) 小鼠免疫實驗 13 (三十四) 血球凝集抑制試驗 14 (三十五) 統計分析 14 第三章 結果 15 (一) 基因的構築、鑑定以及序列分析比對 15 (二) Anti-H1抗體的製備 15 (三) HA蛋白表現在細胞膜上 15 (四) HA蛋白可被釋出到胞外培養基中 15 (五) 不同MOI對於胞外HA產量的影響 16 (六) 磁攪拌培養瓶培養對於HA蛋白產量的影響 17 (七) 以蔗糖梯度離心分離胞外HA蛋白 17 (八) 桿狀病毒在各分層中的分布 18 (九) 以穿透式電子顯微鏡證實H1-VLPs形成 19 (十) 以二次純化方式提升H1-VLPs純度 19 (十一) HA似病毒顆粒的組成分析 20 (十二) 免疫小鼠中的HI抗體力價 20 第四章 討論 21 (一) 評估 A/PR8/34 HA第 237 號胺基酸突變對於似病毒顆粒 產生的影響 21 (二) MOI 對於 HA 似病毒顆粒產量的影響 21 (三) 桿狀病毒表現系統的 HA 釋放到胞外的效率 22 (四) H1 與 H7 HA 似病毒顆粒的比較 22 (五) 似病毒顆粒分層含有其他雜質 22 (六) 其他提高似病毒顆粒純度及產量的方法 23 (七) 似病毒顆粒的蛋白組成 23 參考文獻 25 表 31 圖 35 附錄 66zh_TW
dc.subjectVirus-like particlesen_US
dc.titleProduction of Immunogenic Baculovirus-Derived H1-subtype Influenza Hemagglutinin Virus-Like Particlesen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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