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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳慧文(Hui-Wen Chen) | |
| dc.contributor.author | Wan-Zhen Zhu | en |
| dc.contributor.author | 朱婉蓁 | zh_TW |
| dc.date.accessioned | 2021-06-17T08:07:20Z | - |
| dc.date.available | 2024-08-22 | |
| dc.date.copyright | 2019-08-22 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73645 | - |
| dc.description.abstract | H6亞型禽流感病毒 (AIV) 感染遍佈全世界，並且在台灣雞場中流行40多年，H6 AIV適應的物種較其他亞型廣，在台灣曾發生人與犬隻感染H6 AIV的病例，為了預防可能的大流行，我們須要安全且有效的疫苗作為防控措施。本研究旨在製備H6 AIV雞隻分離株之類病毒顆粒 (VLP) 抗原、混合佐劑作為疫苗，以雞隻動物模式評估疫苗之免疫原性及保護效果。首先將H6 AIV的血球凝集素 (HA) 與基質蛋白1 (M1) 基因選殖至桿狀病毒之表現載體中，接著將重組桿狀病毒感染昆蟲細胞產製VLP，並以蔗糖密度梯度離心純化VLP；經由奈米粒子追踪分析、電子顯微鏡及免疫金染色進行檢測後，可觀察到 VLP的粒徑、表面電位、形態及抗原性都與原態病毒顆粒相似。動物實驗則進一步顯示，VLP混合市售佐劑ISA 71 VG或自製CpG奈米粒子佐劑作為疫苗，能誘發SPF雞隻產生具H6N1 AIV特異性之血清IgG和血球凝集抑制抗體，並維持至少112天；在感染H6N1病毒後，亦降低了免疫組雞隻在淚液與泄殖腔之排毒，以及肺臟與腎臟的病毒複製情形。此外，疫苗在雞隻所誘發的抗體對於H6N1 AIV之人類分離株亦有交叉反應。總結來說，本研究所產製之禽流感病毒類病毒顆粒在雞隻具有良好的免疫原性，與佐劑共同使用，更能提升其抗病毒之保護效果，可作為禽流感病毒之候選疫苗。 | zh_TW |
| dc.description.abstract | H6 avian influenza viruses (AIVs) have a worldwide distribution, and they pose a potential concern for public health. In Taiwan, H6 AIVs have circulated in domestic chickens for more than 40 years, and some of these strains have crossed the species barrier to infect mammals. Towards containing the disease, there is a pressing need to develop a safe and effective vaccine for pandemic preparedness. In this study, we prepared a virus-like particle (VLP) that consists of the hemagglutinin (HA) and matrix protein 1 (M1) derived from an H6 AIV as a vaccine antigen, and its immunogenicity and protective efficacy when combining with adjuvants were examined in a chicken model. Full-length HA and M1 protein genes were cloned and expressed using a baculovirus expression system, and VLPs were purified from the supernatant of the insect cell cultures. Nanoparticle tracking analysis and transmission electron microscopy were performed to validate that the particle’s structure and properties resembled the native virions. In addition, polymeric nanoparticles encapsulating the CpG molecules were prepared as an adjuvant. In animal experiments, SPF chickens receiving the H6 VLPs in combination with ISA 71 VG, a commercial adjuvant, or CpG-encapsulating nanoparticles showed superior H6N1 virus-specific serum IgG and hemagglutination inhibition antibody response, which lasted more than 112 days. Following H6N1 viral challenge, the vaccinated chickens showed significantly reduced viral shedding and tissue viral load. Moreover, antibody induced in chickens by the vaccine can cross-react with the H6 AIV human isolate. In summary, the H6 VLP vaccine elicited superb immunogenicity in vivo and the use of adjuvant further enhanced the antiviral protective efficacy. This vaccine formulation can potentially be used to manage H6 influenza virus infections in chickens. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T08:07:20Z (GMT). No. of bitstreams: 1 ntu-108-R06629004-1.pdf: 1816081 bytes, checksum: 315f616a86214f9a2ed51d92ad430859 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | Contents
誌謝 I 中文摘要 II Abstract IV Chapter 1 Introduction 1 1.1 Influenza virus 1 1.2 Hemagglutinin of influenza virus 2 1.3 Matrix protein of influenza virus 2 1.4 Replication of influenza virus 3 1.5 Pathogenicity of influenza virus 5 1.6 Immune responses of host 7 1.7 Epidemiology of H6 AIVs 8 1.8 Avian influenza virus vaccines 9 1.9 Virus-like particle (VLPs) 10 1.10 ISA 71 VG adjuvant 10 1.11 CpG adjuvant 11 1.12 Nanoparticle system 13 1.13 Objectives of this study 14 Chapter 2 Materials and methods 15 2.1 Cell cultures 15 2.2 Influenza genes and the expression construct 15 2.3 Plaque assays 16 2.4 Optimization of infection condition of recombinant baculovirus 16 2.5 Production and purification of VLPs 17 2.6 Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blot 18 2.7 Propagation and purification of influenza viruses 18 2.8 50% egg infective dose (EID50) titration of influenza virus 20 2.9 Antiserum production 20 2.10 Bradford protein quantification assay 20 2.11 Nanoparticle tracking analysis 21 2.12 Transmission electronic microscopy (TEM) and immunogold staining 21 2.13 Hemagglutination (HA) test 22 2.14 Hemagglutination inhibition (HI) test 23 2.15 Preparation of CpG nanoparticles (NPs) 23 2.16 Chicken immunization and viral challenge studies 24 2.17 Enzyme-linked immunosorbent assay (ELISA) 25 2.18 Viral RNA extraction, reverse transcription and quantitative PCR (qPCR) 26 2.19 Virus re-isolation in embryo eggs 27 2.20 Statistical analyses 27 Chapter 3 Results 28 3.1 Production and purification of H6 VLPs 28 3.2 Characterization of H6 VLPs 29 3.3 Immunogenicity of H6 VLPs with ISA 71 VG 30 3.4 Preparation and characterization of CpG NP 32 3.5 Immunogenicity of H6 VLP with CpG 32 3.6 Protective efficacy of H6 VLP vaccine 33 Chapter 4 Discussion 35 Chapter 5 Figures and tables 42 Chapter 6 References 56 | |
| dc.language.iso | en | |
| dc.subject | H6亞型禽流感病毒 | zh_TW |
| dc.subject | 疫苗 | zh_TW |
| dc.subject | 奈米粒子 | zh_TW |
| dc.subject | CpG | zh_TW |
| dc.subject | 佐劑 | zh_TW |
| dc.subject | 類病毒顆粒 | zh_TW |
| dc.subject | nanoparticle | en |
| dc.subject | adjuvant | en |
| dc.subject | vaccine | en |
| dc.subject | CpG | en |
| dc.subject | H6 Avian influenza virus | en |
| dc.subject | virus-like particle | en |
| dc.title | 類病毒顆粒混合佐劑作為雞隻禽流感病毒疫苗之效果 | zh_TW |
| dc.title | Anti-avian influenza efficacy of a virus-like particle and adjuvant in chickens | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王金和(Ching-Ho Wang),徐維莉(Wei-Li Hsu),陳怡寧(Yi-Ning Chen),胡哲銘(Che-Ming Jack Hu) | |
| dc.subject.keyword | H6亞型禽流感病毒,類病毒顆粒,佐劑,CpG,奈米粒子,疫苗, | zh_TW |
| dc.subject.keyword | H6 Avian influenza virus,virus-like particle,adjuvant,CpG,nanoparticle,vaccine, | en |
| dc.relation.page | 64 | |
| dc.identifier.doi | 10.6342/NTU201903928 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-19 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-108-1.pdf 未授權公開取用 | 1.77 MB | Adobe PDF |
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