請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32231完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張鑫 | |
| dc.contributor.author | Pei-Yu Chu | en |
| dc.contributor.author | 朱珮瑜 | zh_TW |
| dc.date.accessioned | 2021-06-13T03:37:56Z | - |
| dc.date.available | 2007-08-03 | |
| dc.date.copyright | 2006-08-03 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-26 | |
| dc.identifier.citation | 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32231 | - |
| dc.description.abstract | C 型肝炎病毒 (hepatitis C virus, HCV) 為非 A 非 B 型肝炎的主要致病原，與慢性肝炎、肝硬化及肝細胞癌的形成密切相關。HCV 為一正向單股 RNA 病毒，基因體全長約 9600 個核苷酸，包含 5' 端非轉譯區 (noncoding region, NCR)、可轉譯出約 3010 個胺基酸多蛋白質之開放編閱架構、以及 3' 端非轉譯區，其中 5' 端非轉譯區在各個不同病毒分離株之間具有高度保守性。HCV 的轉譯起始是由 internal ribosome entry site (IRES) 所調控，其範圍幾乎涵蓋整個 5' 端非轉譯區，並延伸至轉譯起始點後核心蛋白質的前 10 個胺基酸相對序列。IRES 可形成穩定的二級和三級結構，在功能上取代許多轉譯起始因子，能直接與 40S 核醣體次單元、真核細胞轉譯起始因子 Ⅲ (eukaryotic translation initiation factor 3, eIF3)，以及其他許多細胞因子進行交互作用。然而，目前對於 HCV 內部起始轉譯的詳細機制仍不完全清楚。
真核細胞轉譯起始因子 Ⅲ 為一 800 kDa 之巨大複合體，包含至少 13 個不同的次單元，其中次單元 p170、p116、p66 及 p47會與 C 型肝炎病毒的 IRES 有專一性的結合。根據二級結構預測，p116 在靠近氨基端 (a.a. 185-268) 具有一個可能的 RNA 結合區 (RNA recognition motif, RRM)，可和HCV IRES 結合。過去的研究顯示 p116-RRM 與IRES domain Ⅲ (nt 134-314) 之間有交互作用；但亦有研究指出 p116 會與 domain Ⅱ (nt 44-118) 結合。根據本實驗室先前的研究結果，顯示 domain Ⅱ 的頂端 (nt 65-102) 與domain Ⅲ 中的 abcd subdomains (nt 131-278) 對於 p116-RRM 有相類似的結合能力。因此，p116 在 HCV IRES上的實際結合區域仍有待確認。本研究主要著重在探討 p116-RRM 與 HCV 5' 端 RNA 的結合情形︰藉著將 C型肝炎病毒 5' 端非轉譯區及 core protein coding region 前段序列分割成較小片段，分別與純化的 p116-RRM 進行 filter binding、gel mobility shift、以及UV-crosslinking 等結合測試，試圖找出 p116 在 IRES 上最小的結合區域。結果發現，包含 domain Ⅲab 的 NCR123-232 RNA 即足以與p116-RRM 有很強之結合能力，顯示 p116 與 IRES 的接合位置應位在此區域內。此外，在核心蛋白質轉譯區也偵測到與 p116-RRM 結合的能力。在活體外轉譯實驗中加入涵蓋不同 domain 之 IRES 片段，會對 HCV IRES 所引導之核心蛋白質的合成造成程度不等的抑制效果。對於與 p116-RRM 有較強結合的 RNA 片段，對 IRES 引導的活體外轉譯也具有較強的抑制能力，顯示 C 型肝炎病毒 IRES 與 eIF3 次單元 p116 的結合對於此病毒的轉譯起始是重要的。 | zh_TW |
| dc.description.abstract | Hepatitis C virus (HCV), the major infectious agent of non-A, non-B hepatitis, often causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV is a positive, single-stranded RNA virus with genomic size of approximately 9.6 kb. The viral genome consists of a 5’ noncoding region (NCR), a large open reading frame encoding a polyprotein of approximately 3010 amino acids, and a 3’ NCR. Sequences in the 5’ NCR are highly conserved among HCV isolates. Translation initiation of HCV is mediated by an internal ribosome entry site (IRES) element which encompasses almost the entire 5' NCR and about 30 nt of the core protein coding region immediately downstream the AUG codon. In addition, the IRES folds into a stable secondary and tertiary structure and has been demonstrated to functionally replaces several initiation factors by directly recruiting the 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and other cellular factors. Nevertheless, the mechanism of HCV internal initiation is poorly understood.
Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. eIF3 has an aggregate molecular mass of ~800 kDa and comprises at least 13 subunits. Four subunits of eIF3 complex, p170, p116, p66 and p47 have been demonstrated to specifically bind to the HCV IRES. According to secondary structure prediction, p116 contains a putative RNA recognition motif (RRM) near the N terminal region (a.a. 185-268). Independent studies have demonstrated interactions between p116-RRM and the domain Ⅲ (nt134/314) of HCV IRES, and between the p116 subdomain from amino acid 227 to 320 and the IRES domain Ⅱ (nt 44-118 apical part). In our laboratory, p116-RRM was previously found to interact with both the HCV IRES domain Ⅱ (nt 65-102) and the domain Ⅲabcd (nt 131-278). In this study, the interaction between HCV IRES and the p116 subunit of eIF3 was further examined. His•RRM fusion protein was purified and used to examine its RNA binding ability by filter binding assay, gel shift assay, and UV-crosslinking experiments. The results indicate that IRES domain Ⅲab (nt 123-232) has a stronger RRM-binding activity then the domain Ⅲabcd (nt 131-278) does. Interesting, HCV413-581 that represents a part of the core protein sequence was also found to interact with p116-RRM. By performing in vitro translation competition assay, RNA subdomains important for the HCV-IRES-mediated translation of core protein were examined. RNA subdomains that had stronger ability to interact with p116-RRM shown stronger effect on translation-inhibition when used as a RNA competitor. So the interaction between HCV IRES and p116-RRM has essential effect on the IRES-mediated translation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T03:37:56Z (GMT). No. of bitstreams: 1 ntu-95-R93445108-1.pdf: 2431799 bytes, checksum: c6d6f78bd77a76acfdc1ceee3cdc33d9 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 摘要.…………………………………….....………………… Ⅰ
英文摘要………………………………………………………… Ⅲ 縮寫表…………………………………………………………… Ⅴ 緒論…………………………………………………………… 1 研究方向………………………………………………………… 19 實驗材料與方法………………………………………………… 20 實驗結果………………………………………………………… 36 討論……………………………………………………………… 45 圖表……………………………………………………………… 54 附表與附圖……………………………………………………… 73 參考文獻………………………………………………………… 80 | |
| dc.language.iso | zh-TW | |
| dc.subject | 真核細胞轉譯起始因子 | zh_TW |
| dc.subject | C 型肝炎病毒 | zh_TW |
| dc.subject | 內部核醣體進入位置 | zh_TW |
| dc.subject | eIF3 | en |
| dc.subject | IRES | en |
| dc.subject | HCV | en |
| dc.title | C型肝炎病毒內部核醣體進入位置與真核細胞轉譯起始因子Ⅲ次單元p116交互作用之分析 | zh_TW |
| dc.title | Analysis of the interaaction between HCV IRES and the p116 subunit of eIF3 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳美如,董馨蓮,王萬波 | |
| dc.subject.keyword | C 型肝炎病毒,內部核醣體進入位置,真核細胞轉譯起始因子, | zh_TW |
| dc.subject.keyword | HCV,IRES,eIF3, | en |
| dc.relation.page | 94 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-27 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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