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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張繼堯 | |
dc.contributor.author | "Hsiang-Er, Lee" | en |
dc.contributor.author | 李香爾 | zh_TW |
dc.date.accessioned | 2021-06-08T06:59:55Z | - |
dc.date.copyright | 2011-08-18 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-15 | |
dc.identifier.citation | Reference
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26086 | - |
dc.description.abstract | 石斑魚 (Epinephelus spp.) 為台灣重要的高潛力經濟養殖魚種,由於肉質佳且味道鮮美所以受到廣泛的喜愛。然而石斑從幼魚到成魚時期都會受到虹彩病毒感染威脅且死亡率極高,虹彩病毒不僅感染海水魚類,並且也感染淡水魚類造成相當大的經濟損失,有鑑於此研究石斑魚虹彩病毒便成為重要的課題。本實驗室從石斑魚脾臟分離出虹彩病毒 (grouper iridovirus, GIV),其基因體序列共有 139,793 個鹼基對,推測含有139個開放讀架 (open reading frames, ORFs),以微陣列晶片分析 GIV 基因的表現順序共找出21個極早期表現基因 (Immediate-early gene) 。097L 基因被推測為極早期基因之一,其基因全長 975 個鹼基對,由 325 個胺基酸組成,其構成蛋白分子量為 36.9 kDa 。以 NCBI 資料庫分析 097L 蛋白顯示其碳端含有 ubiquitin E3 ligase 相似序列,且在此序列中含有 RING finger 功能區,因此推測其可能參與蛋白質的降解過程。本實驗首先利用西方墨點法以 097L 抗體証實 097L 蛋白是屬於GIV 病毒顆粒的結構蛋白。而反轉錄聚合酶鏈鎖反應 (RT-PCR) 在虹彩病毒感染後兩小時即可偵測到 097L 的轉錄表現,且以轉譯抑制劑 cycloheximide 處理後,也仍可偵測到轉錄表現,證實 097L 為極早期基因。097L 融合蛋白在 GK 與 HeLa 細胞內的表現,顯示其主要分布在細胞核內少量分布在細胞質中。本實驗利用基因剔除法 (gene knockout) 剔除 097L 基因,並以螢光蛋白作為追蹤標記,顯現 097L 基因剔除的重組病毒相較於野生型病毒在感染 GK 細胞後,有病毒斑 (plaque) 縮小和病毒感染力 (titer) 下降現象,顯示 097L 可能參與虹彩病毒的複製與增殖過程。 | zh_TW |
dc.description.abstract | The grouper (Epinephelus spp.) is an important aquaculture species in Taiwan due to their excellent flavor and high economic value. Recently, high mortalities in grouper caused by iridovirus infection during larvae, juvenils, and adult stage have been resulted in extremely high economical loss. Grouper iridovirus (GIV) had been divided from the spleen of the diseased groupers, and the complete DNA genome had been sequenced in the previous work in our laboratory. The whole 139,793 base pairs contain persumed 139 open reading frames (ORFs), and 21 immediately-early ORFs were identified using microarray method. GIVORF 97L has 975 base pair in length and encodes a protein of 325 amino acids with predicted molecular mass of 36.9 kDa. The I-TASSER analysis showed that the 097L has a RING figer domain located at the C-terminus, which is a critical region of the most E3 ubiquitin ligase involved in protein degradation. The 097L expression can be detected at two hours post infection in GK cells, and it is classified to immediately early gene after cycloheximide examination. The 097L protein is belonged to structure protein of virion component by Western blot analysis. The 097 fusion protein expressed in GK and HeLa cells showed that it’s subcellular localization mainly in nucleus and few in cytoplasm. The EGFP-replaced 097L-knockout recombinant virus was estabolished to investigate the function of 097L. Comparing with the wild type GIV, the 097L-knockout virus created smaller plaque size and owned lower titer. These data revealed that GIV097L could play an important role in virus replication and virus propagation process. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T06:59:55Z (GMT). No. of bitstreams: 1 ntu-100-R97b45014-1.pdf: 2448200 bytes, checksum: e26c2caf12bf2bf26cd955684d4a7769 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Table of content
Abstract ( In Chinese )……………….………………...…………………….…..….…I Abstract ( In English )…….…………………………………….………….……...…II Table of content……….…...……...……………..……………....………..….………IV Figure list………………………………………………………………..…...........….VII Table list………………………………………………………………………….…....IX Appendix list………………………………………………………….…….………......X Chapter 1 Introduction 1 1.1 Background of Taiwan grouper aquaculture 1 1.2 Background of grouper iridovirus 2 1.3 Virion of grouper iridovirus 3 1.4 Immediate early gene of GIV097L 4 1.5 Ubiquitin E3 Ligase 5 1.6 Application of EGFP protein 6 1.7 GIV recombinant virus with 097L gene delection 7 Chapter 2 Materials and Methods 8 2.1 Cell cultures 14 2.2 Grouper iridovirus (GIV) purification 14 2.3 Sequence analysis 15 2.4 Inhibition of protein synthesis and replication activity 15 2.5 GIV097L RNA expression of different post-infection time in GK cells 16 2.6 Subcellular localization of EGFP/GIV097L fusion protein in cells. 17 2.6.1 Construction of pEGFP-N/GIV097L plasmid 17 2.6.2 Transfection of pEGFP-N/GIV097L into GK and HeLa cells 18 2.7 Expression of fusion protein in GK and HeLa cells 18 2.7.1 Construction of pcDNA3CF/GIV097L plasmid 18 2.7.2 Tolerance test of G418 concentration in GK cells 19 2.7.3 Expression of 097L-FLAG and 097L-EGFP fusion protein in cells 20 2.7.4 Western blot analysis of GIV097L recombinant protein 20 2.7.5 Immunocytochemical staining 21 2.8 Generation of a GIV097L knockout recombinant GIV 22 2.8.1 Construction of a knockout pDsRed/GIV097L plasmid 22 2.8.2 Transfection of pDsRed/GIV097L into GK cells 23 2.8.3 Production of GIV097L knockout-EGFP virus by homologous recombination 24 2.8.4 Flow cytometric analysis 24 2.8.5 Freeze-Thaw 25 2.8.6 Purification of recombinant virus by plaque assay and limited dilution 25 Chapter 3 Results 27 3.1 Sequence analysis 27 3.2 Temporal expression pattern of 097L 28 3.3 Virion component of GIV 28 3.4 Localization of GIV097L-EGFP and GIV097L-FLAG fusion protein in GK and HeLa cells 29 3.5 Production of 097 knockout recombinant GIV 30 Chapter 4 Discussion 32 Reference .......................................................................................................................37 Figures………………………...……………………….............................................…50 Tables…………………………………………………………………………….…….55Appendix………………………………………………………………………………75 Figure List Figure 3.1 Diagram of the 097L gene position in grouper iridovirus genome. 48 Figure 3.2 Sequence alignment of GIV097L gene with that of other iridovirus. 49 Figure 3.3 Sequence analysis of the GIV097L gene. 50 Figure 3.4 Temporal expression pattern of GIV097L in GIV-infected GK cells. 51 Figure 3.5 One dimension electrophoresis separation of the GIV proteome. 52 Figure 3.6 Construction of pEGFP-N/GIV097L. 53 Figure 3.7 Expression of GIV097L-EGFP fusion protein in GK cells. 54 Figure 3.8 Expression of GIV097L-EGFP fusion protein in HeLa cells. 55 Figure 3.9 Subcellular localization of GIV097L-EGFP in HeLa and GK cells. 56 Figure 3.10 Construction of pcDNA3CF/GIV097L. 57 Figure 3.11 Expression of GIV097L-FLAG fusion protein in HeLa cells. 58 Figure 3.12 Subcellular localization of GIV097L-FLAG in HeLa and GK cells. 59 Figure 3.13 Diagram of the construction concept of pDsRed2-1/GIV097Lko for 097L knockout recombinant GIV. 60 Figure 3.14 The flow chart of creating GIV097L knockout recombinant GIV. 61 Figure 3.15 Diagram of the homologous recombinant of GIV097L knockout GIV in GK cells. 62 Figure 3.16 Construction of pDsRed2-1/GIV097Lko plasmid. 63 Figure 3.17 Expression of the EGFP reporter genes in GK cells after homologous recombination. 64 Figure 3.18 Flow cytometric analysis of GIV097L knockout recombinant GIV in GK cells. 65 Figure 3.19 Plaque forming of 097L knockout recombinant GIV in GK cells. 66 Figure 3.20 Purification of 097L knockout recombinant GIV by limited dilution. 68 Table List Table 1 Primers used in this study. 69 Appendix List Appendix 1: The mechmisn of cycloheximide (CHX), aphidicoline (APH), acinomycin D and phosphonoacetic acid (PAA) (Modified from Liu et al., 2005). 70 Appendix 2: Avoidance and exploitation of ubiquitylation by virus (Pickart, C. M. 2004). 71 Appendix 3: Figure 1.1 The ubiquitin-proteasome system (Eriksson et al., 1996). 72 Appendix 4: The relationship between the targeting frequency and the length homologous in a replacement vector (Joyner, A. 1993). 73 Appendix 5: SGIV protein identified by 1-DE-MALDI (Song et al., 2006). 74 Appendix 6: Vector Map of pcDNA3CF. 75 Appendix 7: Vector Map of pDsRed2-1. 76 Appendix 8: Vector Map of pEGFP-N1 77 Appendix 9: Vector Map of pECFP-1. 78 | |
dc.language.iso | en | |
dc.title | 石斑魚虹彩病毒極早期基因097L之分子特性表現及其功能之分析 | zh_TW |
dc.title | Molecular characterization, expression and functional assay of grouper iridovirus immediate-early gene 097L | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇建國,林正輝 | |
dc.subject.keyword | 虹彩病毒,極早期基因, | zh_TW |
dc.subject.keyword | iridovirus,immediate early gene, | en |
dc.relation.page | 78 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2011-08-15 | |
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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