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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳秀男(Shiu-Nan Chen) | |
| dc.contributor.author | Chong-Rong Lu | en |
| dc.contributor.author | 呂重融 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:59:01Z | - |
| dc.date.copyright | 2011-08-22 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23309 | - |
| dc.description.abstract | 歸屬虹彩病毒科(Iridoviridae)中細胞腫大病毒屬(Megalocytivirus)的病毒為導致亞洲國家淡水和海水魚類大量死亡的主要因子之一。傳染性脾腎壞死病毒(Infectious spleen and kidney necrosis virus； ISKNV)為細胞腫大病毒屬(Megalocytivirus)之典型種(type species)。ISKNV ORF111L轉譯一個296胺基酸蛋白質，具部分類似於人(Homo sapiens)和家鼷鼠(Mus musculus)的腫瘤壞死因子受體相關因子(TRAF)蛋白質。本篇歸類一株持續感染嘉鱲魚鰭細胞株(PI-RSBF-2)之病毒分離株為ISKNV，並藉由選殖ISKNV ORF111L基因及功能分析來探討病毒蛋白所涉及之細胞凋亡過程或細胞增殖調控。選殖的基因序列與ISKNV ORF111L序列有100% 相似度，其推演之胺基酸片段與其他細胞腫大病毒(Megalocytiviruses) TRAF homolog有高相似度(60%-92%)。ISKNV ORF111L胺基酸序列含有一個TRAF superfamily domain及一個RING finger domain但不具有穿膜片段。系統發生分析結果顯示ISKNV ORF111L與其他細胞腫大病毒TRAF homologs位於同一單元類群且接近魚類TRAF homologs之類群。此外，ISKNV ORF111L的轉錄作用最早於ISKNV感染嘉鱲魚鰭細胞(RSBF-2) 2小時後可偵測到，而RSBF-2細胞過度表現ISKNV ORF111L蛋白質導致星型孢菌素(Staurosporine)誘導之細胞凋亡現象提前產生。細胞生長實驗結果顯示過度表現ISKNV ORF111L蛋白質促使細胞增殖能力提高。 | zh_TW |
| dc.description.abstract | Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. ISKNV ORF111L encodes a 296-residue protein that has some similarity to the tumor necrosis factor receptor-associated factor (TRAF) proteins of Homo sapiens and Mus musculus. In this study, a virus isolated from a persistently infected red sea bream fin cell line (PI-RSBF-2) was classified to ISKNV, and we elucidate the viral protein which involved in apoptosis process or cell proliferarion regulation by cloning ISKNV ORF111L and functional assays. Cloned gene sequence revealed 100% identity to ISKNV ORF111L sequence and the deduced amino acid sequences showed high similarity (60-92%) to other Megalocytiviruses TRAF homolog. ISKNV ORF111L contains a TRAF superfamily domain and a RING finger domain but has no transmembrane fragments. The results from phylogenetic analysis shows that ISKNV ORF111L and TRAF homologs from other Megalocytiviruses are in a monophyletic group and this group is relative close to that from fish TRAF homologs. Furthermore, the transcription of ISKNV ORF111L was detected as early as 2 h post infection in ISKNV infected red sea bream fin cells (RSBF-2). Whereas overexpression of ISKNV ORF111L resulted in advanced apoptosis induced by Staurosporine. In cell growth experiment showed that overexpression of ISKNV ORF111L can promote the proliferation RSBF-2 cells. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:59:01Z (GMT). No. of bitstreams: 1 ntu-100-R98b45031-1.pdf: 6709781 bytes, checksum: 21d68bd6e8dbebeba0bc9b11ab2ce0f1 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 目錄...................................................................................................................................i
中文摘要………………………………………………………….…………....…..........v 英文摘要..........................................................................................................................vi 第一章 文獻整理……………………………………………………….…....................1 1.1 虹彩病毒簡介以及分類地位............................................................................1 1.2 亞洲地區細胞腫大病毒屬(Megalocytivirus)之研究……………………3 1.3 病毒與宿主免疫反應........................................................................................4 1.3.1 宿主抗病毒反應.....................................................................................4 1.3.2 受病毒感染細胞誘導之細胞凋亡.........................................................5 1.3.3 病毒基因產物影響宿主細胞凋亡.........................................................7 1.4 病毒分離及RSBF-2細胞株...............................................................................8 1.5 細胞腫大病毒腫瘤壞死因子受體相關因子(TRAF)基因功能預測................9 1.6 Staurosporine誘導之細胞凋亡.........................................................................11 1.7 研究目的..........................................................................................................12 第二章 材料與方法.......................................................................................................13 2.1 病毒與細胞培養..............................................................................................13 2.1.1 嘉鱲魚鰭細胞株(RSBF-2)繼代培養...................................13 2.1.2 嘉鱲魚鰭細胞株(RSBF-2)冷凍保存...................................13 2.1.3 細胞腫大病毒(Megalocytiviruses)增殖................................13 2.1.4 細胞腫大病毒(Megalocytiviruses)力價測定..............................14 2.2 基因選殖、胺基酸序列比對與分析………………………………………..15 2.2.1 細胞腫大病毒(Megalocytiviruses)去氧核醣核酸(DNA)萃取.…15 2.2.2 病毒Major capsid protein (MCP)基因之聚合酶連鎖反應(PCR)..15 2.2.3 ISKNV-111L基因片段之聚合酶連鎖反應(PCR)……………………16 2.2.4 膠體純化(Gel elution)……………………………………………......16 2.2.5 接合作用(DNA Ligation)…………………………………………….16 2.2.6 大腸桿菌(Escherichia coli, E.coli)勝任細胞製作…………………...17 2.2.7 質體轉型作用(Transformation)………………………………………17 2.2.8 質體純化 (Plasmid extraction)……………………………………….17 2.2.9 限制酶切割(Restriction enzyme digestion)…………………………..17 2.2.10 電腦軟體比對與分析……………………………………………….18 2.3 ISKNV-111L之轉錄(Transcription)分析…………………………………….18 2.3.1 核醣核酸(Total RNA)萃取…………………………………………...18 2.3.2 反轉錄聚合酶鏈鎖反應 (RT-PCR) …………………………………19 2.4 ISKNV-111L 基因功能分析………………………………………………...20 2.4.1 pcDNA3.1-111L質體構築……………………………………………20 2.4.2 細胞轉染(Transfection) ……………………………………………...20 2.4.3 穩定轉染細胞(Stable transfectants)之篩選………………………….20 2.4.4 Staurosporine誘發RSBF-2細胞產生凋亡現象……………………..21 2.4.5 DNA片段化(Fragmentation)分析…………………………………….21 2.4.6 ISKNV-111L基因表現對細胞凋亡之影響…………………………..21 2.4.7 細胞增殖能力分析…………………………………………………...22 第三章 實驗結果……………………………………………………………………...23 3.1 細胞腫大病毒(Megalocytiviruses)分離株(Isolate)鑑定…………………….23 3.2 傳染性脾腎壞死病毒(ISKNV) ORF111L核酸序列選殖及確認…………..23 3.3 ISKNV-111L核酸序列比對………………………………………………….24 3.4 ISKNV-111L胺基酸序列分析與比對……………………………………….24 3.5 ISKNV-111L胺基酸序列系統發生分析…………………………………….25 3.6 ISKNV-111L之轉錄(Transcription)分析…………………………………….26 3.7 pcDNA3.1-111L質體構築……………………………………………………26 3.8 藥物處理誘導細胞凋亡之分析……………………………………………..26 3.9 ISKNV-ORF111L表現對RSBF-2細胞增殖之影響………………………..27 第四章 討論…………………………………………………………………………...28 參考文獻……………………………………………………………………………….34 表目錄 表1……………………………………………………………………………………..49 表2……………………………………………………………………………………..49 圖目錄 圖1：1.5%瓊脂膠體電泳圖………………………………………………………...…50 圖2：確認pGEM-MCP質體構築之0.8%瓊脂膠體電泳圖……………….……......51 圖3：1.5%瓊脂膠體電泳圖………………………………………………………...…52 圖4：確認pGEM-111L質體構築之0.8%瓊脂膠體電泳圖………….…………......53 圖5：ISKNV-111L與多種TRAF homolog胺基酸序列之比對圖……….………....54 圖6：ISKNV-111L之胺基酸序列極性結構圖……………………….…………..….55 圖7：ISKNV-111L與多種TRAF homolog胺基酸序列之系統發生樹………..…..56 圖8：RT-PCR偵測ISKNV-111L轉錄之1.5%瓊脂膠體電泳圖………………......57 圖9：確認pcDNA3.1-111L質體構築之0.8%瓊脂膠體電泳圖……………………58 圖10：Staurosporine誘導RSBF-2細胞凋亡…………………...……………….59 圖11：Staurosporine誘導RSBF/pcDNA3.1-111L細胞凋亡…………………..60 圖12：細胞生長曲線圖…………………………………………………………..61 附錄.................................................................................................................................62 儀器........................................................................................................................62 藥品試劑................................................................................................................63 生物反應試劑組....................................................................................................65 實驗載體................................................................................................................66 | |
| dc.language.iso | zh-TW | |
| dc.subject | 腫瘤壞死因子受體相關因子(TRAF) | zh_TW |
| dc.subject | 星形孢菌素(Staurosporine) | zh_TW |
| dc.subject | 魚鰭細胞株(RSBF-2) | zh_TW |
| dc.subject | 嘉鱲 | zh_TW |
| dc.subject | 細胞凋亡 | zh_TW |
| dc.subject | 傳染性脾腎壞死病毒(ISKNV) | zh_TW |
| dc.subject | Infectious spleen and kidney necrosis virus | en |
| dc.subject | Staurosporine | en |
| dc.subject | RSBF-2 | en |
| dc.subject | apoptosis | en |
| dc.subject | tumor necrosis factor receptor-associated factor | en |
| dc.title | 海水魚類細胞腫大病毒腫瘤壞死因子受體相關因子(TRAF)基因特性分析 | zh_TW |
| dc.title | Characterization of tumor necrosis factor receptor-associated factor gene from megalocytivirus isolated in marine fish | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃世鈴(Shi-Ling huang),王俊順(Chun-Shun Wang) | |
| dc.subject.keyword | 傳染性脾腎壞死病毒(ISKNV),腫瘤壞死因子受體相關因子(TRAF),細胞凋亡,嘉鱲,魚鰭細胞株(RSBF-2),星形孢菌素(Staurosporine), | zh_TW |
| dc.subject.keyword | Infectious spleen and kidney necrosis virus,tumor necrosis factor receptor-associated factor,apoptosis,RSBF-2,Staurosporine, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2011-08-19 | |
| 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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