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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 杜宜殷 | zh_TW |
dc.contributor.advisor | Yi-Yin Do | en |
dc.contributor.author | 林政谷 | zh_TW |
dc.contributor.author | Cheng-Ku Lin | en |
dc.date.accessioned | 2021-07-11T15:19:45Z | - |
dc.date.available | 2024-06-19 | - |
dc.date.copyright | 2019-06-24 | - |
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/123456789/78793 | - |
dc.description.abstract | 植物作為生產次單位口服疫苗之生物反應器,已於不同植物材料間發展出多種生產策略,突破既有之微生物或動物表達醫藥用蛋白質模式,能快速獲得穩定及高品質之醫藥用蛋白質。本論文以植物病毒載體 (viral-based vectors) 暫時性感染、穩定性核基因轉殖 (nuclear transformation) 及葉綠體基因轉殖 (chloroplast transformation) 等三種策略,於不同植物表達重組豬生殖和呼吸綜合症病毒 (porcine reproductive and respiratory syndrome virus, PRRSV) 之次單位疫苗,包括封套醣蛋白 (envelope glycoprotein 5, GP5)、膜蛋白 (membrane protein, M) 與具佐劑功能之大腸桿菌 (Escherichia coli) 熱不穩定毒素B次單位 (heat-labile toxin B subunit, LT-B)。植物病毒載體選用蘭花之齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV),首先分析ORSV 鞘蛋白 (coat protein, CP) 次基因組核醣核酸 (subgenomic RNA, sgRNA) 啟動子活性,顯示CP sgRNA啟動子位於轉譯起始點-290至+18核苷酸 (nucleotide, nt) 間,啟動子短至8 bp即具轉錄活性,-24至+18 nt具有最高的表達量,推測為ORSV CP啟動子核心區域。應用農桿菌針筒注射滲入法 (syringe agroinfiltration) 及真空抽氣滲入法 (vacuum agroinfiltration) 於蝴蝶蘭 (Phalaenopsis) 花瓣、邊沁菸草 (Nicotiana benthamiana) 及其他植物葉片中進行ORSV表達載體暫時性表達,顯示於ORSV cDNA 3’端構築含D型肝炎 (hepatitis delta virus, HDV) 或錘頭型 (hammerhead) 核醣核酸酵素 (ribozyme) 序列,皆能提升GP5蛋白質表現量,佔總可溶性蛋白 (total soluble protein, TSP) 0.043%~0.063%。觀察穩定性核基因轉殖之香蕉植株,生長及外表性狀正常,果實可自然後熟並產生香氣,其表達GP5蛋白質含量約佔0.074% TSP。進行葉綠體轉殖則使用披衣藻 (Chlamydomonas reinhardtii) 表達次單位疫苗,根據披衣藻葉綠體密碼子使用偏性 (codon usage),構築得優化LT-B/ORF6/ORF5融合基因表達質體,以基因槍轉殖法轉殖於披衣藻葉綠體中,經抗生素篩選取得轉殖系後,鑑定蛋白質顯示次單位融合蛋白質成功於轉殖披衣藻中表達,其蛋白質表達量約佔0.065% TSP。 | zh_TW |
dc.description.abstract | Application of plants as a bioreactor for production of subunit oral vaccine has been successfully developed in different plant species to obtain stable and high-quality pharmaceutical proteins. In this study, fused of envelope glycoprotein (GP5), membrane protein (M) of porcine reproductive and respiratory syndrome virus (PRRSV) and heat-labile toxin B subunit (LT-B) of Escherichia coli were expressed in different plants by three strategies: transient expression by viral-based vectors, stable nuclear transformation, and chloroplast transformation. Deletion analysis of Odontoglossum ringspot virus (ORSV) coat protein subgenomic RNA (sgRNA) promoter activity suggested that the minimal CP sgRNA promoter is located between -290 to +18 relative to translation start site (TSS). As short as 8 bp upstream to TSS was sufficient to direct the expression of reporter genes. The -24~+18 around TSS of CP was with the highest expression level and could be the core promoter. ORSV-based viral vectors and revised versions were transformed into petals of Phalaenopsis and leaves of Nicotiana benthamiana by syringe agroinfiltration or vacuum agroinfiltration for transient expression. Addition of hepatitis delta virus (HDV) or hammerhead ribozyme sequences increased at the 3’-end of ORSV cDNA vector containing the expression of GP5 protein from 0.43% to 0.063% of total soluble protein (TSP). Growth and phenotype of nuclear transgenic banana were normal and protein contents of GP5 expressed in transgenic banana fruits about 0.074% of TSP. For chloroplast tramsformation, codons in LT-B/ORF6/ORF5 fused gene were optimized according to the codon usage of Chlamydomonas reinhardtii chloroplast gene bias and transferred into the chloroplast of C. reinhardtii by particle bombardment. The GP5 protein expressed in transgenic C. reinhardtii lines were sequenced and quantified as about 0.065% of TSP after selection by antibiotics. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:19:45Z (GMT). No. of bitstreams: 1 ntu-108-D00628004-1.pdf: 4660657 bytes, checksum: 7452efc2583b8c862909bdded100bec8 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 目錄
摘要 i Abstract iii 圖 ix 表 xi 前言 1 第一章 齒舌蘭輪斑病毒鞘蛋白次基因組核醣核酸之啟動子活性分析 3 1. 1. 前人研究 3 1. 1. 1. 次基因組RNA啟動子之簡介 4 1. 1. 2. 齒舌蘭輪斑病毒之生理生化特性 4 1. 1. 3. ORSV表達質體及相關研究概況 5 1. 2. 材料與方法 6 1. 2. 1. 試驗菌種與植物材料 6 1. 2. 2. 聚合酶連鎖反應 7 1. 2. 3. 大腸桿菌勝任細胞製備 8 1. 2. 4. 膠體內 DNA 之回收與接合反應 8 1. 2. 5. 質體DNA之轉形 9 1. 2. 6. 質體DNA小量製備 9 1. 2. 7. 質體DNA定序 10 1. 2. 8. 基因槍暫時性表達 10 1. 2. 9. 農桿菌電穿孔轉形 11 1. 2. 10. 農桿菌注射感染分析 12 1. 2. 11. 植物GFP觀察與定量 13 1. 2. 12. GUS活性組織化學染色 13 1. 2. 13. 植物蛋白質表達分析 14 1. 3. 結果 15 1. 3. 1. ORSV CP sgRNA啟動子於蝴蝶蘭花瓣暫時性表達分析 15 1. 3. 2. ORSV CP sgRNA啟動子於邊沁菸草暫時性表達分析 16 1. 3. 3. 邊沁菸草暫時性表達之GUS蛋白質定量 16 1. 4. 討論 17 第二章 齒舌蘭輪斑病毒載體表達PRRSV抗原蛋白之研究 27 2. 1. 前人研究 27 2. 1. 1. 重組醫藥用蛋白質生產系統 28 2. 1. 2. 植物病毒載體表達外源重組蛋白之應用 30 2. 1. 3. 豬生殖和呼吸綜合症之簡介 31 2. 1. 4. 以植物生產PRRSV GP5次單位口服疫苗之概況 34 2. 2. 材料與方法 37 2. 2. 1. 試驗菌種與植物材料 37 2. 2. 2. 試驗方法 38 2. 3. 結果 41 2. 3. 1. ORSV基因組於蝴蝶蘭與邊沁菸草葉片之暫時性表達分析 41 2. 3. 2. PRRSV GP5於邊沁菸草葉片暫時性表達分析 42 2. 3. 3. PRRSV ORF6-ORF5與LTB-ORF6-ORF5於菸草葉片之暫時性 表達分析 42 2. 4. 討論 43 第三章齒舌蘭輪斑病毒表達質體之修整強化 57 3. 1. 前人研究 57 3. 2. 材料與方法 58 3. 2. 1. 試驗菌種與植物材料 58 3. 2. 2. PRRSV GP5蛋白質表達質體之修整構築 58 3. 2. 3. 農桿菌真空抽氣感染法 59 3. 2. 4. 蛋白質免疫呈色分析 59 3. 3. 結果 60 3. 3. 1. 修整強化之質體於邊沁菸草暫時性表達分析 60 3. 3. 2. 修整強化質體於邊沁菸草暫時性表達之GP5蛋白質分析 60 3. 3. 3. 蝴蝶蘭花瓣農桿菌真空抽氣滲入測試與蛋白質定量 61 3. 3. 4. 萵苣與蕃薯葉真空抽氣滲入測試 61 3. 4. 討論 61 第四章 PRRSV GP5短縮蛋白質於大腸桿菌表達 76 4. 1. 前人研究 76 4. 2. 材料與方法 78 4. 2. 1. 試驗菌種與材料來源 78 4. 2. 2. 試驗方法 78 4. 3. 結果 81 4. 3. 1. GP5短縮蛋白質理化特性分析 81 4. 3. 2. 不同溫度與時間條件誘導GP5短縮蛋白質表達 82 4. 3. 3. ORF5短縮蛋白質切膠純化法 82 4. 4. 討論 82 第五章 表達PRRSV GP5次單位疫苗香蕉轉殖株果實之分析 89 5. 1. 前人研究 89 5. 2. 材料與方法 91 5. 2. 1. 植物材料 91 5. 2. 2. GUS化學活性組織染色 91 5. 2. 3. 香蕉果實之可溶性蛋白質萃取 92 5. 2. 4. 蛋白質分析試驗方法 92 5. 3. 結果 92 5. 3. 1. 表達PRRSV GP5香蕉轉殖株果實與植株外表性狀 92 5. 3. 2. 表達PRRSV GP5香蕉轉殖株之GUS活性組織化學染色分析 93 5. 3. 3. 表達PRRSV GP5香蕉轉殖株不同部位組織之蛋白質分析 93 5. 4. 討論 94 第六章 披衣藻葉綠體表達PRRSV GP5抗原蛋白之研究 101 6. 1. 前人研究 101 6. 1. 1. 葉綠體基因轉殖 102 6. 1. 2. 藻類生物反應器培養系統簡介 103 6. 1. 3. 披衣藻葉綠體基因轉殖概況 104 6. 1. 4. 基因密碼子優化 106 6. 1. 5. 基因密碼子優化設計之理論 107 6. 1. 6. 密碼子最適化設計之方法 109 6. 2. 材料與方法 109 6. 2. 1. 試驗菌種與植物材料 109 6. 2. 2. 試驗方法 110 6. 2. 3. 利用基因槍轉殖法進行披衣藻葉綠體基因轉殖 111 6. 2. 4. 葉綠體擬轉殖株之分子驗證 112 6. 3. 結果 115 6. 3. 1. 披衣藻類葉綠體同義密碼子偏性之LTB/ORF6/ORF5序列 分析 115 6. 3. 2. 披衣藻類葉綠體同義密碼子偏性優化之LTB/ORF6/ORF5 胺基酸分析 115 6. 3. 3. 披衣藻葉綠體表達LTB/ORF6/ORF5基因擬轉殖系分子檢測 116 6. 3. 4. 披衣藻葉綠體表達LTB/ORF6/ORF5基因之蛋白質檢測與 鑑定 116 6. 4. 討論 117 綜合討論與未來展望 137 參考文獻 141 附錄. 本論文選殖、質體構築及分析使用之引子 180 | - |
dc.language.iso | zh_TW | - |
dc.title | 植物生產外源蛋白質之研究 | zh_TW |
dc.title | Studies on Expression of Heterologous Proteins in Plants | en |
dc.type | Thesis | - |
dc.date.schoolyear | 107-2 | - |
dc.description.degree | 博士 | - |
dc.contributor.coadvisor | 黃鵬林 | zh_TW |
dc.contributor.coadvisor | Pung-Ling Huang | en |
dc.contributor.oralexamcommittee | 莊榮輝;鄭謙仁;李昆達 | zh_TW |
dc.contributor.oralexamcommittee | Rong-Huay Juang;Chian-Ren Jeng;Kung-Ta Lee | en |
dc.subject.keyword | 植物次單位口服疫苗,齒舌蘭輪斑病毒,植物病毒載體,葉綠體基因轉殖,披衣藻,密碼子優化,豬生殖和呼吸綜合症, | zh_TW |
dc.subject.keyword | plant subunit oral vaccines,Odontoglossum ringspot virus,plant viral-based vectors,chloroplast transformation,Chlamydomonas reinhardtii,codon optimization,porcine reproductive and respiratory syndrome, | en |
dc.relation.page | 181 | - |
dc.identifier.doi | 10.6342/NTU201900883 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2019-06-19 | - |
dc.contributor.author-college | 生物資源暨農學院 | - |
dc.contributor.author-dept | 園藝暨景觀學系 | - |
dc.date.embargo-lift | 2024-06-24 | - |
顯示於系所單位: | 園藝暨景觀學系 |
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