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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張明富(Ming-Fu Chang) | |
dc.contributor.author | Hai-Ning Lin | en |
dc.contributor.author | 林海寧 | zh_TW |
dc.date.accessioned | 2021-06-13T06:58:03Z | - |
dc.date.available | 2016-10-05 | |
dc.date.copyright | 2011-10-05 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-22 | |
dc.identifier.citation | Abrahem, A., and M. Pelchat. 2008. Formation of an RNA polymerase II preinitiation complex on an RNA promoter derived from the hepatitis delta virus RNA genome. Nucleic Acids Res 36:5201-11.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35551 | - |
dc.description.abstract | D型肝炎病毒(hepatitis delta virus, HDV)是目前已知最小的動物病毒,其基因體為負向單股環狀的RNA,全長約為1.7 kb。D型肝炎病毒是一種缺陷型的病毒(defective virus),必須獲得B型肝炎病毒的外套膜才具有感染力,因此被歸類為B型肝炎病毒的衛星病毒。D型肝炎病毒可轉譯出兩種抗原,小型delta抗原(small delta antigen, HDAg-S;195個胺基酸,約24 kDa)參與病毒基因體之複製,而大型delta抗原(large delta antigen, HDAg-L;214個胺基酸,約27 kDa)則與病毒顆粒之組裝有關。
關於D 型肝炎病的的複製機制仍有許多尚未清楚之處。許多文獻指出D型肝炎病毒的基因股及反基因股RNA 複製可能是透過不同的宿主聚合酶來達成。目前已知基因股RNA 的複製是利用宿主第二型RNA聚合酶(DNA-dependent RNA polymerase II)來進行。然而,反基因股RNA 的合成則須仰賴α-amanitin resistent 的聚合酶來負責。先前本實驗室研究發現小型delta 抗原與核仁素(nucleolin)的結合對於小型delta 抗原分佈於核仁及D型肝炎病毒的複製是重要的。且小型delta 抗原的中間區段第89 至163 個胺基酸會與第一型RNA聚合酶(DNA-dependent RNA polymerase I)之最大次單元RPA194 有交互作用,並且RPA194 與D 型肝炎病毒之基因股RNA 可以被共同沈澱下來。除此之外,大量表達小型delta 抗原會抑制宿主rRNA 新生成。這些證據皆指向第一型RNA聚合酶可能參與D 型肝炎病毒反基因股RNA 的合成。 本研究擬探討小型delta 抗原與第一型RNA 聚合酶調控D 型肝炎病毒複製的分子機制。首先建構帶有TAT-domain 與小型delta 抗原各結構區的蛋白質表現質體。TAT-domain 可攜帶其它蛋白質藉由轉導作用進入細胞,並執行其生理功能。將質體經轉型作用送入大腸桿菌株C43(DE3)中進行蛋白質表現,再以親合性管柱層析及透析方式可成功純化出帶有小型delta 抗原中間區段(第89 至163個胺基酸)的TAT 融合蛋白質,TAT-HDAg-S-I。將TAT-HDAg-S-I 融合白質直接加入細胞培養液後可於Hun7 細胞中被偵測到。進一步以間接免疫螢光染色及親和力結合實驗分析發現,無論在in vitro 或in vivo 情形下,TAT-HDAg-S-I 融合蛋白質與細胞中之RNA polymerase I 皆存在交互作用。另一方面,由聚合酶鏈鎖反應分析發現,TAT-HDAg-S-I 能支持D 型肝炎病毒反基因股RNA 的複製,同時減少細胞中rRNA 的新合成。而利用核醣核蛋白質複合體免疫沉澱法分析顯示,在處理TAT-HDAg-S-I 融合蛋白質情況下會增加與RNA polymerase I 共同沉澱下來之病毒RNA。綜合以上結果推論,小型delta 抗原中間區段能與RNA polymeraseI 結合,並且此交互作用能增加其與病毒RNA 的結合,進而促進D 型肝炎病毒的複製,同時此作用會使rRNA 之新合成受到抑制,進而可能影響細胞的正常生長功能。 | zh_TW |
dc.description.abstract | Hepatitis delta virus (HDV) is the smallest known animal virus, with a single-stranded, negative-polarity circular RNA genome of approximately 1.7 kb in length. HDV is considered as a satellite virus of hepatitis B virus (HBV) because it requires the HBV surface antigen (HBsAg) for virion assembly and transmission. HDV contains only one gene, which encodes for two isoforms of hepatitis delta antigen (HDAg). The small delta antigen (HDAg-S, 195 amino acids, 24 kDa) is necessary for the HDV RNA replication, while the large delta antigen (HDAg-L, 214 amino acids, 27 kDa) is critical for the viral assembly.
The detailed mechanisms of HDV replication are still unknown. However, accumulating evidence indicated that the synthesis of genomic and antigenomic RNAs involved two independent transcription machineries: DNA-dependent RNA polymerase II mediates the synthesis of HDV genomic RNA and mRNA, and an α-amanitin resistant RNA polymerase activity is required for the synthesis of antigenomic RNA. Our previous studies demonstrated that the interaction between HDAg-S and nucleolin is important for the nucleolus targeting of HDAg-S and HDV replication. In addition, the middle domain (a.a. 88-163) of HDAg-S interacted with the large subunit of DNA-dependent RNA polymerase I, RPA194. RPA194 could be co-precipitated with HDV antigenomic RNA. Furthermore, overexpression of HDAg-S inhibited the de novo synthesis of rRNA. These results imply that DNA-dependent RNA polymerase I may participate in the synthesis of HDV antigenomic RNA. In this study, molecular mechanisms of HDAg-S involved in the regulation of the RNA polymerase I-associated RNA synthesis were further examined. Expression plasmid encoding TAT-fused subdomains of HDAg-S were generated and expressed in E. coli C43(DE3). TAT-HDAg-S-I representing TAT fusion protein of HDAg-S with amino acid residues 89-163 that specifically interacted with DNA-dependent RNA polymerase I was partially purified by affinity chromatography and rapid dialysis. TAT domain can directly drive protein cargoes into the cells to perform their biological functions. When added into the culture medium, purified TAT-HDAg-S-I protein was readily detected in Huh7 cells. Immunofluorescence and affinity binding assays showed that TAT-HDAg-S-I associated with RNA polymerase I in vitro as well as in vivo. In addition, results from RT real-time PCR analysis indicated that TAT-HDAg-S-I increased the level of HDV antigenomic RNA but reduced the level of rRNA. Furthermore, ribonucleoprotein immunoprecipitation assay demonstrated an enhanced association between RNA polymerase I and HDV RNA when cells were treated with TAT-HDAg-S-I. Taken together, these results suggest that the middle domain of HDAg-S can help the synthesis of HDV antigenomic RNA by recruiting RNA polymerase I to the viral RNA, and meanwhile affects host cell survival through inhibiting rRNA synthesis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:58:03Z (GMT). No. of bitstreams: 1 ntu-100-R98442005-1.pdf: 1752559 bytes, checksum: fae931c882d81f2d7824679b702307e8 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 中文摘要..................................................Ⅰ
英文摘要..................................................Ⅲ 縮寫表....................................................Ⅴ 緒論...................................................... 1 實驗材料................................................. 14 實驗方法................................................. 20 實驗結果................................................. 40 討論..................................................... 47 圖表..................................................... 52 參考文獻................................................. 71 | |
dc.language.iso | zh-TW | |
dc.title | D型肝炎病毒小型delta抗原之中間區段與DNA依賴性RNA聚合酶I之交互作用在病毒複製中扮演的角色 | zh_TW |
dc.title | Roles of the interaction between the middle domain of small delta antigen and DNA-dependent RNA polymerase I involved in the viral replication | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),余明俊(Ming-Jiun Yu),廖經倫(Ching-Len Liao) | |
dc.subject.keyword | D型肝炎病毒,小型delta抗原,DNA依賴性RNA聚合酶,I, | zh_TW |
dc.subject.keyword | HDV,small delta antigen,DNA-dependent RNA polymerase I, | en |
dc.relation.page | 79 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-22 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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