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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 王金和 | |
| dc.contributor.author | You-Hsin Lin | en |
| dc.contributor.author | 林祐新 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:22:33Z | - |
| dc.date.available | 2013-08-20 | |
| dc.date.copyright | 2012-08-20 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-16 | |
| dc.identifier.citation | 第六章、參考資料
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63903 | - |
| dc.description.abstract | 中文摘要
於台灣所分離到的家禽傳染性支氣管炎病毒毒株當中也有數個毒株經過神經胺酸酶的處理後會表現出血球凝集 (Hemagglutination, HA)的特性。其中一台灣一型家禽傳染性支氣管炎病毒2575-5在經過神經胺酸酶處理過後會表現出血球凝集的特性,但該毒株之減毒株在神經胺酸酶處理後則不會表現出血球凝集的特性,為瞭解此血球凝集特性之改變,本實驗將減毒株之S1蛋白基因進行選殖,另將2575-5的S1蛋白基因進行點突變,並將此點突變與選殖之產物放入pFastBacHT A質體中,接著利用同源重組將在pFastBacHT A內的S1醣蛋白轉移至bacmid上並轉染Sf-9細胞產生重組之桿狀病毒,以桿狀病毒蛋白表現系統進行表現蛋白,經過繼代三代後以蔗糖梯度離心純化重組之病毒之後進行血球凝集與血球吸附試驗,經由此實驗可以表現出所欲研究之S1蛋白並且將兩者進行比較來了解該蛋白上之特定胺基酸改變是否與其血液凝集特性改變有關,且可瞭解所表現之蛋白是否與傳染性支氣管炎之S1蛋白有相同之生物特性。 | zh_TW |
| dc.description.abstract | Abstract
A Taiwan local infectious bronchitis virus strain 2575 TW-I showed hemagglutination activity after neuraminidase treatment while its attenuated strain did not. In order to investigate this altered hemagglutination activity, we cloned the S1 gene of the attenuated strain. The nucleotide differences between original and attenuated strains were mutated in the S1 gene by site direct mutation. The S1 gene in the pFastBacHT A was transferred to the bacmids in DH10Bac. The Bac-to-Bac baculovirus expressing system was used to express both wild and attenuated strain S1 proteins. After transfection and passages, the P3 stock of recombinant baculovirus was generated and purified by sucrose gradient separation. The hemagglutination (HA) activity and cell-associtaed hemadsorption (Hd) activity of the expression proteins were examined. Both recombinant baculoviruses showed Hd but no HA activities. The Hd activities can be inhibited by anti-IBV antiserum. Through this method, desired S1 protein mutant could be expressed with the baculovirus expressing system and further studied for biological activity and immunological reactivity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:22:33Z (GMT). No. of bitstreams: 1 ntu-101-R99629007-1.pdf: 2193949 bytes, checksum: 2d1a8c9f05e489f693fdd8bdc0f8d4f2 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書……………………………………………………………………...# 致謝……………………………………………………………………………………i 摘要…………………………………………………………………...………………ii Abstract……………………………………………………………………………….iii 目錄…………………………………………………………………………...………iv 表次……………………………………………………………………………….….vii 圖次…………………………………………………………………………..……...viii 第一章 序言…………………………………………………………………………..1 第二章 文獻回顧……………………………………………………………………..3 第一節 歷史背景.................................................................................................3 第二節 病毒分類.................................................................................................4 第三節 病毒之結構與功能............................ ....................................................5 2-3.1 病毒型態.............................................................................................5 2-3.2 病毒結構.............................................................................................6 2-3.2.1 IBV之病毒基因.........................................................................6 2-3.2.2棘突醣蛋白.................................................................................7 2-3.2.3封套蛋白.....................................................................................9 2-3.2.4 基質醣蛋白................................................................................9 2-3.2.5核殼蛋白.....................................................................................9 第四節 病原性與致病機製................................................................................10 2.4.1自然宿主與傳播途徑...................................................................10 2.4.2病毒傳播........................................................................................11 2.4.3病毒複製........................................................................................11 2.4.4臨床症狀與病灶...........................................................................12 第五節 病原鑑定................................................................................................13 2.5.1病毒分離.......................................................................................13 2.5.2抗原捕捉法...................................................................................14 2.5.3核酸診斷.......................................................................................14 第六節 預防與控制............................................................................................15 2.6.1活毒疫苗.......................................................................................16 2.6.2死毒疫苗.......................................................................................17 2.6.3次單位疫苗...................................................................................18 第七節 昆蟲桿狀病毒與桿狀病毒蛋白質表現系統........................................18 第三章 材料與方法....................................................................................................22 3.1 病毒株與細胞株...........................................................................................22 3.2 RNA萃取.......................................................................................................22 3.3增幅台灣一型傳染性支氣管炎病毒2575-75之S1基因...........................23 3.4點突變............................................................................................................23 3.5 傳染性支氣管炎病毒2575-75全段S1基因與點突變質體之選殖..........24 3.6 構築重組bacmid DNA.................................................................................25 3.7 轉染與重組病毒之增殖...............................................................................26 3.8 測定重組病毒力價.......................................................................................26 3.9 純化重組病毒...............................................................................................27 3.10表現重組S1蛋白........................................................................................27 3.11純化重組S1蛋白........................................................................................27 3.12重組病毒與純化後重組蛋白之蛋白質定量..............................................28 3.13以西方墨點法偵測重組S1蛋白................................................................28 3.14以免疫螢光染色法確認重組蛋白之表現..................................................30 3.15以間接型酵素連結免疫吸附試驗 (Indirect ELISA)測定重組蛋白與各型別血清之反應.....................................................................................................31 3.16測定重組蛋白之血球吸附試驗.................................................................31 3.17利用帶有禽流感病毒HA蛋白之腺病毒感染細胞進行之血球吸附試驗.........................................................................................................................32 3.18以各型別之血清針對重組病毒進行血球吸附抑制試驗.........................32 3.19以重組桿狀病毒進行血球凝集抑制試驗.................................................33 3.20重組病毒之血球凝集試驗.........................................................................33 3.21 IBV結構蛋白之醣基去除與血球凝集反應.............................................34 3.22 統計分析....................................................................................................34 第四章 結果 4.1 Bacmid DNA之重組與點突變....................................................................35 4.2重組病毒之構築、定量與純化...................................................................35 4.3重組病毒之表現與純化...............................................................................36 4.4重組S1蛋白與重組桿狀病毒之抗原性.....................................................36 4.5血球凝集試驗與血球吸附試驗...................................................................37 4.6血球吸附與血球凝集抑制試驗...................................................................38 4.7醣基去除對血球凝集之影響.......................................................................39 第五章 討論...............................................................................................................40 第六章 未來展望.......................................................................................................46 第七章參考資料.........................................................................................................47 附錄.............................................................................................................................78 | |
| dc.language.iso | zh-TW | |
| dc.subject | 血球凝集 | zh_TW |
| dc.subject | 傳染性支氣管炎 | zh_TW |
| dc.subject | 血球吸附 | zh_TW |
| dc.subject | Hemadsorption | en |
| dc.subject | Hemagglutination | en |
| dc.subject | Infectious bronchitis virus | en |
| dc.title | 傳染性支氣管炎病毒S1醣蛋白質與血球吸附、血球凝集關係之探討 | zh_TW |
| dc.title | Hemagglutination and hemadsorption activities associated with S1 glycoprotein of infectious bronchitis virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭益謙,闕玲玲,陳秋麟 | |
| dc.subject.keyword | 血球吸附,血球凝集,傳染性支氣管炎, | zh_TW |
| dc.subject.keyword | Hemadsorption,Hemagglutination,Infectious bronchitis virus, | en |
| dc.relation.page | 84 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-16 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| Appears in Collections: | 獸醫學系 | |
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| File | Size | Format | |
|---|---|---|---|
| ntu-101-1.pdf Restricted Access | 2.14 MB | Adobe PDF |
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