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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | zh_TW |
| dc.contributor.author | 呂芝瑩 | zh_TW |
| dc.contributor.author | Zhi-Ying Lu | en |
| dc.date.accessioned | 2021-07-10T21:54:23Z | - |
| dc.date.available | 2024-08-12 | - |
| dc.date.copyright | 2019-08-14 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77289 | - |
| dc.description.abstract | H7N9流感病毒自2013年爆發後，至2018年為止共有6波流感疫情，平均死亡率約為40%，且病毒株已突變為高病原性，並逐漸適應人群，對人類健康的潛在威脅更高，因此開發H7N9流感病毒的快速檢驗平台與治療性抗體日益重要。本實驗室已製備出抗H7N9流感病毒血球凝集素之鼠源單株抗體F3-2，此論文進一步選殖出F3-2之VH與VL基因，並透過大腸桿菌表現出可溶形式的F3-2單鏈抗體，且其具有結合H7N9 virion的能力。每1 L之大腸桿菌約可表現及純化出2 mg之F3-2單鏈抗體。為了使單鏈抗體在原態時能形成雙聚體，使其在結合能力上更接近單株抗體，本研究亦改變了單鏈抗體中連結VL與VH間之peptide linker的長度。當peptide linker少於5個胺基酸時，可以形成二價的diScFv，並且仍保有結合H7N9 virion的能力。每1 L之大腸桿菌約可表現及純化出1.63 mg之F3-2 diScFv。F3-2 diScFv與ScFv對H7HA之解離常數介於12.9–88.9 nM之間。本論文亦於F3-2單鏈抗體之C端接上抗病毒胜肽 (F3-2 ScFv-AVP)，希望可以增加其抑制H7N9流感病毒之功效。然而，F3-2 ScFv-AVP雖保有結合H7N9 virion的能力，且對H7HA的解離常數介於5.7–54.4 nM之間，但仍無法有效抑制H7N9流感病毒感染MDCK細胞。 | zh_TW |
| dc.description.abstract | After the first outbreak of the H7N9 influenza virus in 2013, there were six waves of H7N9 influenza epidemics until 2018, with an average mortality rate of about 40%. The virus strain has been mutated to high pathogenicity and gradually adapted to the population, posing a potential threat to human health. The preparedness of rapid-test kits and neutralizing antibodies against the novel H7N9 virus is becoming more and more important. F3-2 is a mouse monoclonal antibody developed in our laboratory that specifically targets H7N9 HA. The present study further cloned the VH and VL genes of F3-2 and expressed the recombinant F3-2 ScFv (single chain variable fragments) in E. coli as the soluble form, which retained the ability for binding to H7N9 virion. 2 mg of F3-2 ScFv can be purified from 1 L of E. coli culture. In order to make ScFvs form a dimer in the native condition to mimic the antigen-binding capability of a monoclonal antibody, a peptide linker with different length was also inserted between VL and VH domains. Data showed that when the peptide length was shorter than five amino acid residues (e.g. GGS, GGGS, or GGGGS), the F3-2 ScFv mainly formed as a diScFv, which also contained the ability for binding to H7N9 virion. 1.63 mg of F3-2 diScFv can be purified from 1 L of E. coli culture. The dissociation constants (KD) of F3-2 ScFv and F3-2 diScFv for binding to H7HA were in the range of 12.9-88.9 nM. To increase the capability of F3-2 ScFv and F3-2 diScFv for neutralizing H7N9 influenza virus, the antiviral peptide (AVP) was added to the C-terminus of the F3-2 ScFvs. The F3-2 ScFv-AVP performed the ability for binding to H7N9 virion, and had KD in the range of 5.7-54.4 nM for binding to H7HA. However, F3-2 ScFv-AVP couldn’t neutralize H7N9 virus replication in MDCK cells. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:54:23Z (GMT). No. of bitstreams: 1 ntu-108-R06b22051-1.pdf: 8417596 bytes, checksum: dafff9e5655a0a7a99a007dc3e9ca06e (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄
摘要 i Abstract ii 縮寫表 iii 第一章 緒論 1 1.1 A型流感病毒血球凝集素 1 1.1.1 A型流感病毒血球凝集素之分類 1 1.1.2 血球凝集素之結構與功能 1 1.1.3 血球凝集素之抗原位點 2 1.2 抗體之結構與功能 3 1.3 單鏈抗體 4 1.3.1 單鏈抗體之生化性質 5 1.3.2 抗血球凝集素之中和性抗體 5 1.3.3 針對不同亞型之血球凝集素的中和性抗體 6 1.4 抗病毒胜肽 7 1.4.1 抗病毒胜肽之生化性質 7 1.4.2 抑制血球凝集素功能之抗病毒胜肽 7 1.5 H7N9流感病毒 8 1.6 抗H7N9流感病毒血球凝集素之鼠源單株抗體F3-2 9 1.7 研究動機 9 第二章 材料與方法 11 2.1 單株抗體F3-2之VH與VL的基因選殖 11 2.2 建構F3-2 ScFv表現質體 11 2.3 建構F3-2 ScFv diabody (diScFv) 12 2.4 建構F3-2 ScFv-AVP與F3-2 diScFv-AVP 12 2.5 以大腸桿菌表現重組抗體 12 2.5.1 表現F3-2 ScFv與diScFv 12 2.5.2 表現F3-2 ScFv-AVP與diScFv-AVP 13 2.6 Bradford蛋白質定量法 13 2.7 以親和性層析法純化重組抗體 13 2.8 以膠體過濾法分析抗體分子大小 14 2.9 H7N9 HA重組蛋白的表現與純化 14 2.10 測定重組抗體對抗原之解離常數 15 2.11 聚丙烯醯胺凝膠電泳 16 2.12 CBR染色法 16 2.13 西方墨點法 16 2.14 酵素免疫吸附分析法 17 2.15 MDCK細胞培養基配方、病毒培養基配方 17 2.16 病毒增殖 18 2.16.1 活毒病毒，使用在病毒中和試驗中 18 2.16.2 死毒病毒，濃純病毒，使用在ELISA和WB實驗中 18 2.17 抗體對病毒之中和性測試 19 第三章 結果 21 3.1 F3-2抗體基因選殖 21 3.2 F3-2單鏈抗體建構 21 3.3 F3-2 ScFv-20之蛋白質表現與純化 21 3.4 F3-2 ScFv-20保有結合H7N9 virion之功能 22 3.5 F3-2 diScFv的建構 22 3.6 F3-2 diScFv之蛋白質表現與純化 23 3.7 F3-2 ScFv VL與VH間之peptide linker長度與ScFv能否形成diScFv有關 24 3.8 F3-2 ScFv與F3-2 diScFv對於H7N9 virion之結合力分析 24 3.9 F3-2 ScFv與F3-2 diScFv對於H7N9 HA之解離常數分析 25 3.10 F3-2 ScFv-AVP與F3-2 diScFv-AVP的質體建構 25 3.11 F3-2 ScFv-AVP與F3-2 diScFv-AVP之蛋白質表現與純化 26 3.12 F3-2 ScFv-AVP與F3-2 diScFv-AVP維持其聚體特性 27 3.13 F3-2 ScFv-AVP與F3-2 diScFv-AVP對於H7N9 virion之結合力分析 28 3.14 F3-2 ScFv-AVP與F3-2 diScFv-AVP對於H7N9 HA之解離常數分析 28 3.15 F3-2 diScFv-AVP在細胞實驗中不具有中和H7N9病毒的能力 28 第四章 討論 31 參考文獻 33 圖與表 39 附錄 57 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 抗病毒胜? | zh_TW |
| dc.subject | H7N9 流感病毒 | zh_TW |
| dc.subject | 血球凝集素 | zh_TW |
| dc.subject | 單鏈抗體 | zh_TW |
| dc.subject | the H7N9 influenza virus | en |
| dc.subject | antiviral peptides | en |
| dc.subject | single chain variable fragments | en |
| dc.subject | hemagglutinin | en |
| dc.title | 抗 H7N9 流感病毒血球凝集素之單鏈抗體的製備與生化性質分析 | zh_TW |
| dc.title | Preparation and Biochemical Characterization of the Anti-H7N9 Influenza Virus Hemagglutinin ScFv Antibodies | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 廖憶純;陳慧文;游傑華 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | H7N9 流感病毒,血球凝集素,單鏈抗體,抗病毒胜?, | zh_TW |
| dc.subject.keyword | the H7N9 influenza virus,hemagglutinin,single chain variable fragments,antiviral peptides, | en |
| dc.relation.page | 59 | - |
| dc.identifier.doi | 10.6342/NTU201902854 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-11 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| 顯示於系所單位： | 生化科技學系 | |
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