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標題: | 嚴重急性呼吸道症候群冠狀病毒核殼蛋白質負調控 Daxx 蛋白質之轉錄抑制作用 SARS-CoV nucleocapsid protein deregulates transcriptional repression of death-associated protein |
作者: | Chun-Kai Huang 黃俊凱 |
指導教授: | 張明富 |
關鍵字: | 嚴重急性呼吸道症候群,冠狀病毒,核殼蛋白質, SARS-CoV,nucleocapsid,Daxx,death-associated protein, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 嚴重急性呼吸道症候群相關之冠狀病毒 (severe acute respiratory syndrome associated coronavirus,SARS-CoV) 被證實與引起 SARS 之非典型肺炎有關,其致死率約 9.6 %。SARS-CoV 為一含有套膜之新型冠狀病毒,基因體為一正向的 RNA,長約 30,000 個核苷酸,具有 14 個 ORFs,其中包括四個結構性蛋白質,用以組成病毒顆粒,分別為 spike (S)、membrane (M)、envelope (E),及 nucleocapsid (N)。本實驗研究對象是 SARS-CoV 之 nucleocapsid 蛋白質,其一般功能是負責病毒 RNA 之組合。
本實驗室先前利用高效率酵母菌功能性基因模組篩選出一個細胞因子 death-associated protein (Daxx) 會與 N 蛋白質產生交互作用。目前 Daxx 已知功能在細胞質參與強化 CD95 (Fas) 所誘導之細胞凋亡及在細胞核中參與轉錄抑制。為了更進一步確立 N 與 Daxx 的交互作用,首先以免疫共同沉澱 (coimmunoprecipitation) 的方式證明,在哺乳類動物細胞中 N 的確會與 Daxx 發生活體內 (in vivo) 交互作用,且其結合位置落在 Daxx1-625 蛋白質片段中,為 sumoylation-independent。此外利用共軛焦顯微鏡觀察,發現當大量表現 Daxx 時,會使得 N 與 Daxx 共位 (colocalization) 在細胞核的比例上升。SARS-N 可負調控 Daxx 對 IL-8 promoter 的轉錄抑制作用。進一步實驗證明,N 可誘導 IL-8 promoter 之活化,此活化現象會受大量表現 Daxx 所抑制。推測 N 可能藉由與 Daxx 的結合負調節 Daxx 在基因轉錄抑制之調控。 除此之外,本論文也嘗試建立重組腺病毒表現系統來表達 N 蛋白質。藉由此一系統的建立,未來將可以在感染的細胞及動物模式中探討 SARS-N 蛋白質之致病機轉。 Severe acute respiratory syndrome (SARS) is an atypical pneumonia caused by SARS-coronavirus (SARS-CoV). The overall mortality rate was 9.6 %. SARS-CoV is an enveloped, positive single-stranded RNA virus with an RNA genome about 30,000 nucleotides in length, which encodes 14 open reading frames (ORFs). The virus particle consists of four structural proteins, spike (S), membrane (M), envelope (E), and nucleocapsid protein (N). By performing high throughput yeast functional array analysis, our laboratory has previously identified death-associated protein (Daxx) as a SARS-CoV N-interacting protein. Daxx is known to strengthen CD95 (Fas)-induced apoptosis pathway in cytoplasm. Nevertheless, when localized to the nucleus, Daxx can also function as a transcription repressor that inhibits expression of its downstream genes via interacting with transcription factors. The interaction between N and Daxx protein was further examined by co-immunoprecipitation assay. Co-immunoprecipitation assay mapped a specific interaction domain between the N and Daxx protein from amino acid residues 1-625 in transfected mammalian cells, indicating that the interaction is sumoylation-independent. In addition, confocal microscopy detected a significant increase of the colocalization of SARS-N and Daxx in the nucleus when Daxx protein was overexpressed. Furthermore, SARS-N deregulated the transcriptional repression of IL-8 mediated by Daxx protein. SARS-N protein activated the promoter activity of IL-8. The transcriptional activation was inhibited by overexpression of Daxx. Therefore, we proposed that SARS-N protein may modulate Daxx-mediated transcriptional repression by interacting with Daxx protein. On the other hand, a recombinant adenovirus system that expressed the SARS-N protein was established in this study. The infection system may be used to study the pathogenesis of SARS-N protein in cell culture systems and in animal models in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32751 |
全文授權: | 有償授權 |
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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