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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳慧文(Hui-Wen Chen) | |
| dc.contributor.author | Wan-Ting Liao | en |
| dc.contributor.author | 廖婉廷 | zh_TW |
| dc.date.accessioned | 2021-06-17T06:18:14Z | - |
| dc.date.available | 2020-11-13 | |
| dc.date.copyright | 2020-11-13 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-10-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71994 | - |
| dc.description.abstract | 過去幾年，禽流感在家禽健康與公共衛生造成巨大的威脅，並導致大量的經濟損失，由於禽流感病毒的迅速傳播能力、高適應能力及快速變異，野外的野鳥及家禽皆可發現禽流感病毒的蹤影。而最有效的控制疾病方式是透過疫苗施打來達到預防的效果，疫苗中常使用的抗原種類之一為類病毒顆粒 (virus-like particles, VLPs)，因其無法在宿主體內複製並傳播的特性使之成為疫苗研發的目標。在此篇研究中，抗原為台灣及各國常見的H5N6，H6N1及H7N9亞型禽流感病毒之全長血球凝集素(HA)蛋白，及H6N1的基質(M1)蛋白，基因分別選殖在不同桿狀病毒載體上共感染昆蟲細胞所形成的三價類病毒顆粒 (H567-M1 VLPs)，另外是將H5N6，H6N1和H7N9全長HA與牛白血病病毒的gag polyprotein同時選殖在一個桿狀病毒載體上，以此生產出三價類病毒顆粒 (H567-Bgag VLPs)。我們發現利用Sf21細胞表現此些蛋白質的情況比使用HighFive細胞的情況穩定，也發現H567-Bgag VLPs的表現較H567-M1 VLPs穩定；另外使用免疫螢光染色法確定H5/HA, H6/HA及H7/HA皆有被細胞表現；並且利用陰離子交換樹脂進行純化，將桿狀病毒與VLPs分離，但此效果並不顯著。此外在HA活性表現上H567-Bgag VLPs為每25 l 中有8192 HAU，也比H567-M1的64 HAU高；而在形態方面，H567-Bgag VLPs的大小約為145 nm，而H567-M1 VLPs則是91 nm。總結上述結果，H567-Bgag VLPs的表現較H567-M1 VLPs穩定且量多，將作為繼續生產三價類病毒顆粒來進行後續的疫苗試驗，確認其作為抗原的效果。 | zh_TW |
| dc.description.abstract | Avian influenza (AI) has posed a serious threat to the poultry industry and public health, and has resulted in tremendous economic and social impacts in the last few decades. Avian influenza viruses (AIVs) are widespread in domestic and wild birds due to their highly contagious property and frequent adaptive evolution. To prevent and control the AIVs, vaccination is one of the most effective ways. As for the antigens being used in vaccines, virus-like particles (VLPs) are promising candidates due to their native and non-infective natures. Here, we developed a trivalent VLP displaying the hemagglutinin (HA) of H5, H6 and H7 AIVs, the most common AIV subtypes found in Taiwan and other countries. Full-length HA protein genes of AIVs H5N6, H6N1, H7N9 and the matrix protein 1 (M1) gene of AIV H6N1 were cloned onto baculoviral vectors separately, and the trivalent VLPs (H567-M1 VLPs) were then generated by co-infecting cells with three individual recombinant baculoviruses. On the other hand, full length of HA protein genes and gag protein gene from the bovine leukemia virus were cloned onto the baculoviral vector, and the trivalent VLPs (H567-Bgag VLPs) were then generated by infecting insect cells with one single recombinant baculoviruses. The results showed that, use of Spodoptera frugiperda 21 (Sf21) culture for infection improved the expression yield of trivalent VLPs, as compared with the HighFive cells. The expression of H567-Bgag VLPs was more stable than that of H567-M1 VLPs. Furthermore, immunofluorescence assay was used to determine H5/HA, H6/HA and H7/HA expression in Sf21 cells and each subtype were expressed successfully. Then, VLPs were purified from the Sf21 culture supernatant by anion exchange chromatography. The morphology of VLPs was confirm by TEM. The size of H567-M1 VLPs were 91 nm and that of H567-Bgag VLPs’ was 145 nm. The hemagglutination activity of H567-M1 VLPs and H567-Bgag VLPs were 64 HAU per 25 l and 8192 HAU per 25 l after purification. In summary, we developed a versatile VLP platform displaying HA antigens of multiple subtypes. Multivalent VLP antigens with high quality are anticipated to show promises in the application of vaccine development for disease control. | en |
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| dc.description.tableofcontents | Contents 誌謝 ................................................................................................................................ I 中⽂摘要 ...................................................................................................................... II Abstract ........................................................................................................................ IV Chapter 1. Introduction .................................................................................................. 1 1.1 Avian influenza virus ....................................................................................... 1 1.1.1 H5 influenza virus .......................................................................................... 2 1.1.2 H6 influenza virus .......................................................................................... 3 1.1.3 H7 influenza virus .......................................................................................... 4 1.2 Virus structure and components ........................................................................ 5 1.3 Life cycle of influenza virus .............................................................................. 5 1.4 Hemagglutinin of influenza virus ...................................................................... 7 1.4.1 Glycosylation of hemagglutinin ......................................................................... 9 1.5 Matrix protein of influenza virus ..................................................................... 10 1.6 Virus-like particles (VLPs) ............................................................................. 11 1.6.1 VLPs expression systems ............................................................................... 12 1.6.2 Production and purification of baculovirus expression system ............................... 13 1.6.3 Applications of VLPs .................................................................................... 14 1.7 Objectives of this study .................................................................................. 15 Chapter 2. Materials and Methods ............................................................................... 17 2.1 Cell culture .......................................................................................................... 17 2.2 Influenza virus genes (VLP strains) ........................................................................ 17 2.3 Cloning of H5-HA and H567-Bgag genes and the generation of recombinant baculoviruses ............................................................................................................. 18 2.4 Plaque assay ......................................................................................................... 19 2.5 Optimization of expression conditions of H567-M1 VLPs and H567-Bgag VLPs ..... 19 2.6 VLPs purification ................................................................................................. 20 2.7 Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blot ........................................................................................................................... 21 2.8 Bradford protein quantification assay ..................................................................... 22 2.9 Immunofluorescence assay (IFA) ........................................................................... 23 2.10 Hemagglutination (HA) test ................................................................................. 24 2.11 Transmission electronic microscopy (TEM) and dynamic light scattering (DLS) ..... 24 Chapter 3. Results ........................................................................................................ 25 3.1 H5-HA construction and expression ................................................................ 25 3.2 Optimization the infection conditions of H567-M1 VLPs in insect cells ............. 26 3.3 H567-Bgag construction and expression .......................................................... 27 3.4 Optimization the infection condition of H567-Bgag VLPs in insect cells ............ 28 3.5 Purification of H567-M1 VLPs and H567-Bgag VLPs ...................................... 28 3.6 Characterization of H567-M1 VLPs and H567-Bgag VLPs ............................... 29 Chapter 4 Discussion ................................................................................................... 31 Chapter 5 Figures and tables ....................................................................................... 35 Chapter 6 References ................................................................................................... 55 | |
| dc.language.iso | en | |
| dc.subject | 三價類病毒顆粒 | zh_TW |
| dc.subject | H5亞型禽流感病毒 | zh_TW |
| dc.subject | H6亞型禽流感病毒 | zh_TW |
| dc.subject | H7亞型禽流感病毒 | zh_TW |
| dc.subject | 血球凝集素 | zh_TW |
| dc.subject | H5 avian influenza virus | en |
| dc.subject | trivalent virus-like particles | en |
| dc.subject | hemagglutinin | en |
| dc.subject | H7 avian influenza virus | en |
| dc.subject | H6 avian influenza virus | en |
| dc.title | 禽流感H5/H6/H7亞型三價類病毒顆粒之製備與分析 | zh_TW |
| dc.title | Generation and Characterization of H5/H6/H7 Trivalent Avian Influenza Virus-like Particles | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王金和(Ching-Ho Wang),張世宗(Shih-Chung Chang) | |
| dc.subject.keyword | H5亞型禽流感病毒,H6亞型禽流感病毒,H7亞型禽流感病毒,血球凝集素,三價類病毒顆粒, | zh_TW |
| dc.subject.keyword | H5 avian influenza virus,H6 avian influenza virus,H7 avian influenza virus,hemagglutinin,trivalent virus-like particles, | en |
| dc.relation.page | 63 | |
| dc.identifier.doi | 10.6342/NTU202004255 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-10-13 | |
| 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 | |
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