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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張繼堯(Chi-Yao Chang) | |
| dc.contributor.author | Ching-Hui Hsu | en |
| dc.contributor.author | 徐靖惠 | zh_TW |
| dc.date.accessioned | 2021-05-20T20:53:15Z | - |
| dc.date.available | 2016-08-18 | |
| dc.date.available | 2021-05-20T20:53:15Z | - |
| dc.date.copyright | 2011-08-18 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-03 | |
| dc.identifier.citation | 曾文陽。石斑魚養殖學。前程出版社，1998年。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9977 | - |
| dc.description.abstract | 石斑魚(Epinephelus spp.)為台灣重要的經濟養殖魚種之一，卻長期飽受虹彩病毒威脅，使得石斑魚養殖業面臨重大的經濟損害，因此研究虹彩病毒為刻不容緩的課題。石斑魚虹彩病毒(grouper iridovirus, GIV)的基因依據轉錄的先後順序可分為三大類：極早期基因( immediate early gene )、早期基因( early gene )和晚期基因( late gene )；其中，極早期基因能藉由調控病毒基因表現或改變宿主細胞的生理狀態，例如影響宿主細胞生長週期、細胞凋亡以及免疫防禦系統等，促進病毒進行複製增殖，因此極早期基因對於病毒感染宿主具有重要功能。本實驗室分析石斑魚虹彩病毒基因表現次序，推測其具有二十一個極早期基因，其中包含了ORF108L。108L基因全長為1,149個鹼基對，由382個胺基酸組成，蛋白質分子量 44.1 kDa，經由序列分析比對顯示其與 ICP46蛋白相似，且在虹彩病毒科中具有高度的保留性，因此認為108L對於病毒感染應扮演十分重要的角色；然而，目前尚未發現 ICP46具有已知有意義的功能區，所以它的功能仍是未知的。
本實驗建構了原核表現載體pET-28a-CBP-Factor Xa-108L，並成功地在大腸桿菌BL-21(DE3)以IPTG誘導表現出可溶性的重組蛋白。經由反轉錄聚合酶鏈鎖反應(RT-PCR)偵測石斑魚虹彩病毒感染石斑魚腎臟(GK)細胞病毒的基因表現，顯示108L於感染後2小時即開始轉錄，此外以轉錄抑制劑 (cycloheximide) 處理後仍可大量表現，證實108L基因確實屬於病毒極早期基因。藉由免疫細胞化學染色發現無論在GK或HeLa細胞內，108L蛋白表現位置主要為細胞核；此外本實驗基於同源性重組(homologous recombination)概念建構108L基因剔除(108L gene knockout )的重組病毒，並比較野生型石斑魚虹彩病毒與108L基因剔除重組病毒感染石斑魚腎臟細胞的差異性，顯示重組病毒出現效價(titer)下降與病毒斑(plaque)變小之現象。本實驗結果顯示108L可能於細胞核內作用並參與石斑魚虹彩病毒增殖與複製調控。 | zh_TW |
| dc.description.abstract | Grouper (Epinephelus spp.) is an important aquaculture fish species in Taiwan, but it is highly susceptible to iridovirus which often cause significant economic losses to grouper aquaculture. Accordingly, it is imperative to investigate the mechanisms of iridovirus infection and pathogenesis. The grouper iridovirus (GIV) genes can be classed into immediate early (IE), early (E) and late (L) genes according to their temporal synthesis upon infection. IE genes are regard as major roles in virus life cycle, because the transcripts of viral IE genes manipulate essential functions to benefit viral replication including controlling itself gene expression and altering host cell physiological status, such as cell cycle control, apoptosis and immune response. ORF108L is one of the immediate-early genes which our laboratory had identified from GIV. It contains 1,149 nucleotide and is composed of 382 amino acids which encode a 44.1 kDa protein. By comparative sequence analysis, 108L encodes infected cell polypeptide (ICP) 46 homolog which is highly conserved among the Iridoviridae family. However, there are no putative conserved domains have been found in ICP46 protein, so its actual function remains unknown.
The prokaryotic expression plasmid, pET-28a-CBP-Factor Xa-108L, was constructed and transformed into the E.coli strain BL-21 (DE3) for expression. Besides, the best conditions of expression soluble 108L-his recombinant protein and purification by Ni2+ affinity column are well-established. The RT-PCR data confirmed that GIV108L is an immediate early gene of GIV, because the transcript of GIV108L was firstly detected at 2 hours post infection and still expressed after cycloheximide treatment. By immunocytochemistry assay, GIV108L protein was predominantly distributed at the nucleus both in GK and HeLa cells. Finally, the 108L gene knockout virus is generated by homologous recombination. Comparing wild-type virus with recombinant virus, the virus titeration is lower and the presence of cytopathic effect (CPE) obscured by infecting GK cells with recombinant virus than wild-type virus. In summary, this study demonstrated that GIV108L may function in nucleus and involve in the propagation and replication of grouper iridovirus. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-20T20:53:15Z (GMT). No. of bitstreams: 1 ntu-100-R98B45020-1.pdf: 2880870 bytes, checksum: 732d8e357cab9819e6d7edf8e20d2f5b (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 摘要 I
Abstract II 目錄 IV 圖目次 VII 表目次 VIII 目錄 第一章 前言 1 1.1 石斑魚之簡介與台灣養殖概況 1 1.2 虹彩病毒 (Iridovirus) 2 1.2.1 虹彩病毒之分類 2 1.2.2 虹彩病毒之特性 4 1.2.3 石斑魚虹彩病毒極早期基因的發現 5 1.3 病毒極早期基因之功能 5 1.4 病毒之 ICP46 ( Infected-Cell Protein 46) 6 1.5 DNA重組技術 6 1.6 研究動機 7 第二章 材料與方法 9 實驗材料與藥品試劑 9 實驗方法 14 2.1 細胞培養 (cell culture) 14 2.1.1 細胞株及培養條件 14 2.1.2 細胞繼代 (passage) 培養 14 2.2 石斑魚虹彩病毒 (grouper iridovirus, GIV)增殖 14 2.3 質體構築 (construction of plasmids) 15 2.3.1 蛋白表現載體 15 2.3.2 108L基因剔除載體 16 2.4 勝任細胞 (competent cell) 的製備 17 2.5 轉形作用 (Transformation) 17 2.6 細胞轉染 (Transfection) 18 2.7 重組病毒的製備與純化 18 2.7.1重組病毒的產生 18 2.7.2病毒斑試驗 (Plaque assay) 18 2.7.3病毒液序列稀釋 19 2.8 DNA 萃取 19 2.9 RNA萃取 19 2.10 RT-PCR 20 2.11 大腸桿菌重組蛋白之表現 21 2.12 大腸桿菌重組蛋白之純化 21 2.13 SDS-PAGE 電泳 22 2.14 西方墨點法 (Western blot) 22 2.15 免疫螢光染色 22 第三章 結果 24 3.1 GIV108L的序列與特性之分析 24 3.2 GIV108L為極早期基因 24 3.3 以原核表現系統生產GIV108L可溶性重組蛋白 25 3.4 GIV 108L蛋白在細胞內的表現位置與表現量 26 3.4.1 GIV108L融合蛋白於細胞內之表現位置 26 3.4.2 GIV108L融合蛋白於細胞內之表現量 26 3.5 GIV 108KO重組病毒之產生 27 3.6 重組病毒之純化過程 28 3.6.1 病毒斑試驗純化重組病毒 28 3.6.2 流式細胞儀篩選發出綠色螢光的細胞 28 3.6.3 病毒液序列稀釋感染GK細胞 29 3.6.4 重組病毒感染之表現型(phenotype)變化 29 3.7 GIV 108KO重組病毒之效價分析與野生型與重組型病毒之比例變化 29 第四章 討論 31 參考文獻 36 圖目次 圖一、 石斑魚虹彩病毒108L之基因與胺基酸序列 44 圖二、 15種已知序列的虹彩病毒之ICP46胺基酸序列比對分析 46 圖三、 15種已知序列的虹彩病毒之ICP46胺基酸親緣性分析 47 圖四、 石斑魚虹彩病毒108L之蛋白質結構分析 48 圖五、 GK細胞感染GIV病毒後不同時間點與蛋白合成抑制藥劑處理後108L之表現情形 49 圖六、 原核系統表現載體pET-28a-CBP-Factor Xa-108L之建構 50 圖七、 利用大腸桿菌BL-21 (DE3) 表現石斑魚虹彩病毒108L-His 融合蛋白 51 圖八、 利用Ni2+親和性層析管柱純化石斑魚虹彩病毒可溶性108L-His 融合蛋白 52 圖九、 真核細胞表現載體pEGFP-N1-108L之建構 53 圖十、 GIV108L-EGFP融合蛋白在GK與HeLa細胞中的表現位置 55 圖十一、 真核細胞表現載體pcDNA3CF-108L之建構 56 圖十二、 GIV108L-Flag融合蛋白在GK與HeLa細胞中的表現位置 57 圖十三、 GIV108L-EGFP融合蛋白在GK與HeLa細胞內的表現量分析 58 圖十四、 GIV108L-Flag融合蛋白在GK與HeLa細胞中的表現量分析 59 圖十五、 pcDNA-3CF載體於GB與GK兩種石斑魚細胞之轉染效率比較 61 圖十六、 p108KO載體之建構 62 圖十七、 108KO重組病毒之產生 64 圖十八、 病毒斑試驗之108KO重組病毒純化 65 圖十九、 流式細胞儀篩選之108KO重組病毒純化 66 圖二十、 序列稀釋感染之108KO重組病毒純化 68 圖二十一、 108KO重組病毒純化過程之表現型(phenotype)變化 70 圖二十二、 PCR檢測野生型與重組型病毒之比例變化與病毒效價分析 71 表目次 表一、實驗使用之引子序列 72 表二、15種已知序列之虹彩病毒ICP46資料統整 73 | |
| dc.language.iso | zh-TW | |
| dc.title | 石斑魚虹彩病毒108L極早期基因之特性鑑定 | zh_TW |
| dc.title | Identification and characterization of the ORF108L, an immediate-early gene of grouper iridovirus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 陳秀男(Shiu-Nan Chen) | |
| dc.contributor.oralexamcommittee | 林正輝(Cheng-Hui Lin) | |
| dc.subject.keyword | 石斑魚虹彩病毒,極早期基因,ICP46蛋白,基因剔除重組病毒, | zh_TW |
| dc.subject.keyword | grouper iridovirus,immediate early gene,ICP46 protein,gene knockout recombinant virus, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2011-08-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
| 顯示於系所單位： | 漁業科學研究所 | |
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