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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
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dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
dc.contributor.author | Ming-Shou Hsieh | en |
dc.contributor.author | 謝明孝 | zh_TW |
dc.date.accessioned | 2021-06-08T03:43:09Z | - |
dc.date.copyright | 2019-07-02 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-06-10 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21701 | - |
dc.description.abstract | 本實驗室早先篩選得一株可辨認H6HA1禽流感病毒 (A/chicken/Taiwan/ 2838V/00) HA頭部序列的單株抗體EB2,並發現其抗原決定基上第二個鹼性胺基酸Arg-201 (R201) 受到HA1上N167之醣基所保護,可以抵抗來自胰蛋白酶之降解,以維持病毒的感染性。而R201位點之存在,於H6亞型的病毒中乃極度保守。為了探討此位點在感染機制上扮演的角色,我們發展了具有外泌功能的雙效型桿狀病毒表現系統,以表現具有醣基化的H6HA1樣本,與去除穿膜區接近全長的H6HA0樣本。此一表現質體使用GP67信號肽做為外泌序列,可將重組蛋白外泌至無血清培養基中以方便純化。在表現蛋白基因下游帶有共用mRNA之internal ribosome entry site 序列,可於胞內表現綠螢光蛋白質做為報導基因,做為表現蛋白質的偵測訊號。更使用此重組桿狀病毒感染斜紋夜盜蛾幼蟲,收集血淋巴之後,可以純化出醣基化之HA1蛋白質,並確認了此重組HA1具有單體與三聚體兩種形式。從細胞培養與蟲體表現之重組H6HA1可以誘發小鼠產生專一性抗H6亞型之抗血清,以做為次單位疫苗使用。所表現之H6HA1存在為三聚體形式,而H6HA0則只有單元體形式。H6HA1三聚體對胰蛋白酶水解具有較強抗性,但卻不能與胎球蛋白唾液酸受體結合。反之,單體H6HA0不具抵抗胰蛋白酶水解抗性,但卻可與胎球蛋白唾液酸受體結合。我們發現,鹼性胺基酸R201之正電荷對HA與宿主細胞唾液酸受體間的結合極為重要。HA0的R201A點突變重組蛋白,失去了對於胎球蛋白唾液酸受體的結合力。此外,HA與唾液酸受體的結合能力也受到環境pH值影響,也能被幾種帶電荷胺基酸抑制。根據以上觀察結果,我們提出了一種病毒與宿主細胞結合的兩階段模型。首先,流感病毒透過HA1上受體結合位,對宿主細胞表面之唾液酸受體進行專一性辨認,當病毒以胞飲進入入宿主細胞後,接著在胞內endosome的酸性環境下,HA三聚體頭部的R201正電荷可協助HA進行對細胞膜的錨定,並觸發後續的感染融合過程。 | zh_TW |
dc.description.abstract | In our previous study, we produced a monoclonal antibody EB2 that recognized an epitope in the HA1 domain on the hemagglutinin (HA) of H6N1 influenza virus(A/chicken/Taiwan/2838V/00). The residue Arg-201 (R201) on this epitope was protected by the glycan at Asn-167 (N167) from tryptic digestion; therefore, the infectivity of the virus was retained. R201 was extremely conserved in various subtypes of the influenza virus. To explore the role of R201 and the protecting glycan, we developed a bi-cistronic baculovirus expression system for the production of H6HA1 and H6HA0 (nearly full-length HA), which were glycosylated in insect cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, coexpressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype. The expressed H6HA1 was mostly found in the trimeric form, and the H6HA0 protein was only found in the monomeric form. The trimeric H6HA1 was resistant to tryptic digestion; however, it could not bind to fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans. By contrast, the monomeric H6HA0 could bind to fetuin but was sensitive to tryptic digestion. We found that the positive charge on R201 was critical for binding HA to the negatively charged surface of host cells because the mutant R201A of H6HA0 lost its binding capacity substantially. Moreover, this binding capacity was dependent on the pH value and inhibited by free electrically charged amino acids. We propose a two-step model for binding the influenza virus with a host cell. The first step involved the specific recognition of the receptor binding site on HA to the sialylated glycan on the host cell. After the virus is engulfed by the acidic endosome, R201 could bind to the cell surface with stronger interactions and trigger the fusion process. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:43:09Z (GMT). No. of bitstreams: 1 ntu-108-D99642013-1.pdf: 4486369 bytes, checksum: c3409adf130896b2019df249f9b9366c (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 目 錄
致謝 i 目錄 ii 圖表目錄 v 中文摘要 vi 英文摘要 viii 第一章 緒論 1 1.1 禽流感的傳播歷史與起源 1 1.2 禽流感病毒 3 1.3 禽流感病毒的重要蛋白質 4 1.4 禽流感病毒的生活史 8 1.5 禽流感病毒對人類的潛在危險性 9 1.6 醣基化對於調控血球凝集素致病機制扮演重要角色 12 1.7 單株抗體工具 14 1.8 重組蛋白質表現系統 15 1.9 微生物表現系統 16 1.10 桿狀病毒-昆蟲細胞表現系統 17 1.11 研究動機及目的 20 第二章 材料與方法 23 2.1 病毒與抗原蛋白質定量 23 2.2 質體構築 23 2.3 膠體電泳 25 2.4 質體接合 25 2.5 轉型作用 25 2.6 以原核表現系統大量生產重組蛋白質 26 2.7 重組桿狀病毒的製備 27 2.8 重組桿狀病毒的純化 27 2.9 重組桿狀病毒的增殖、感染與力價標定 27 2.10 昆蟲細胞的貼附、懸浮培養與感染 28 2.11 以斜紋夜盜蛾幼蟲生產重組HA 28 2.12 HA結合唾液酸受體能力測試 29 第三章 結果與討論 30 3.1 建構具醣基化之雙效型外泌桿狀病毒表現系統 30 3.2 構築不同外泌之訊號肽比較其外泌效果 32 3.3 重組HA6HA1醣基化之確認與檢定 34 3.4 雙效型外泌桿狀病毒表現系統之表現與純化 36 3.5 利用斜紋夜盜蛾幼蟲生產重組H6HA1 39 3.6 自血淋巴中純化重組H6HA1 41 3.7 重組H6HA1誘導小鼠產生抗血清測試 43 3.8 H6HA1可組裝成三聚體與三聚體之抗trypsin水解保護效果 45 3.9 H6HA1上N167對於R201位點具有保護效果 48 3.10 帶正電荷之鹼性胺基酸R201位點可能參與HA1錨定SA受體 52 3.11 酸鹼值與游離胺基酸對於HA0與胎球蛋白受體結合的影響 56 3.12 流感病毒與宿主受體兩階段結合模型 58 第四章 未來研究方向 60 4.1 N167醣鏈對R201保護現象的延伸驗證 60 4.2 HA三聚體可能存在Tense form與Relax form兩種構形 60 4.3 使用Cyro-EM與smFRET驗證兩種構形 61 4.4 細胞免疫螢光染色觀察HA對宿主細胞受體之結合 62 4.5 深入探討RBS與R201在受體結合角色上之關聯性 62 4.6 撰寫適合比對搜尋病毒序列之專用程式 62 參考文獻 63 附錄A 生化實驗法 71 A.1 蛋白質電泳檢定法 71 A.2 單株抗體之製備 79 附錄B 相關補充數據資料 90 問答錄 99 | |
dc.language.iso | zh-TW | |
dc.title | H6N1禽流感病毒血球凝集素之Arg-201為病毒與宿主細胞結合之重要關鍵 | zh_TW |
dc.title | Arg-201 on hemagglutinin is required for H6N1 avian influenza virus to bind with host cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 劉?睿(Je-Ruei Liu) | |
dc.contributor.oralexamcommittee | 何杰龍(Jie-Long He),張世宗(Shih-Chung Chang),王金和(Ching-Ho Wang),楊健志(Chien-Chih Yang) | |
dc.subject.keyword | 禽流感病毒,胺基酸電荷,受體結合位,兩階段結合模型, | zh_TW |
dc.subject.keyword | Avian influenza virus,Charged amino acid,Receptor binding site,Two-step binding process., | en |
dc.relation.page | 104 | |
dc.identifier.doi | 10.6342/NTU201900809 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2019-06-10 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 生物科技研究所 | zh_TW |
Appears in Collections: | 生物科技研究所 |
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