博碩士論文 90224019 詳細資訊




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姓名 江佩玲(Pei-ling Chiang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 登革熱非結構性蛋白3之核苷酸及核酸三磷酸水解酶活性研究
(Studies of the nucleotide triphosphatase and RNA triphosphatase activities of Dengue virus type II Nonstructural protein 3)
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摘要(中) 登革熱病毒非結構性蛋白3是一個具有多種活性的蛋白,其對病毒複製很重要。此蛋白N端三分之一具有絲胺酸蛋白水解酶活性,而C端三分之二具有解旋酶、核苷三磷酸水解酶及核酸三磷酸水解酶等活性。在黃熱病毒類病毒的非結構性蛋白3的C端三分之二段區域內含有7個高度保守性序列,其中之motif I與motif II與核苷三磷酸水解酶活性有關,但是核酸三磷酸水解酶的活性區位置尚不清楚。本論文中製備第二型登革熱病毒非結構性蛋白3 C端三分之二的重組蛋白,並證實此重組蛋白具核苷三磷酸水解酶及核酸三磷酸水解酶的活性。但是motif I中Lys199Ala及Thr200Ala突變會導致活性喪失。核苷三磷酸水解酶活性需要鎂離子,而核酸三磷酸水解酶活性不需要鎂離子。提高鈉離子濃度對核苷三磷酸水解能力影響不大但會降低核酸三磷酸水解酶活性,亦會減弱核苷三磷酸水解能力被核酸活化的效果。因此核苷三磷酸水解活性及核酸三磷酸水解活性應該位於登革熱病毒非結構性蛋白3相同的活性區,但二水解反應作用機制應不盡相同。
摘要(英) The nonstructural protein NS3 of dengue virus possesses multiple enzymatic activities and is critical to viral replication. The serine protease activity is located in the N-terminal one third of NS3 protein, while the NTPase, helicase, and RNA triphosphatase activities are associated with the remaining 70% of the protein. Seven conserved helicase motifs are found in the C-terminal region of all members of the Flaviviridae family. Motifs I and II of the NS3 protein are directly involved in the NTPase activity, while the functional region of the RNA triphosphatase activity has not been characterized. In this study, a recombinant protein containing the C terminal two thirds of the dengue virus type II NS3 protein was prepared. The wild type protein possessed NTPase activity and RNA triphosphatase activity, while the Lys199Ala and Thr200Ala mutations in the motif I caused the loss of both activities. The NTPase activity is magnesium-dependent, in contrast the RNA triphosphatase activity does not require magnesium. The elevation of NaCl concentration profoundly inhibited the RNA triphosphatase activity and the RNA-stimulated NTPase activity, but had relatively small effect on the NTPase activity. These results indicate that the NTPase activity and the RNA triphosphatase activity are likely to share the same catalytic center, but two hydrolysis reactions may have different molecular mechanism.
關鍵字(中) ★ 核酸三磷酸水解酶
★ 核苷三磷酸水解酶
★ 登革熱
關鍵字(英) ★ dengue
★ NTPase activity
★ RTPase activity
論文目次 英文摘要
中文摘要
目次
表目錄
圖目錄
壹、 引言
貳、 實驗材料與方法
一、實驗材料
二、實驗方法
(一)、登病毒非結構性蛋白3之C 端 (DII3H)表現質體之構築
(二)、登病毒非結構性蛋白3之C 端突變蛋白 (DII3HMu)表現質體之構築
(三)、重組蛋白DII3H及其點突變蛋白質體的轉形 (transformation)
(四)、重組DII3H蛋白及其點突變蛋白質體的表現 (expression)
(五)、SDS-PAGE分析
(六)、重組DII3H蛋白質及其點突變蛋白質的純化及分析
(七)、重組蛋白DII3H之稀釋
(八)、核苷三磷酸水解活性 (NTPase activity)實驗
(九)、放射線標定之核糖核酸 (RNA)之製備
(十)、核酸三磷酸水解活性 (RTPase activity)實驗
(十一)、活性實驗之定量
參、 結果
第二型登革熱NS3之C端重組蛋白DII3H及其突變蛋白質DII3HMu的表現與純化
核苷三磷酸水解活性 (NTPase activity)
影響DII3H的核苷三磷酸水解活性之生物化學因子
DII3H的核苷三磷酸水解活性之Km及Vmax
核酸三磷酸水解活性 (RTPase activity)實驗
影響核酸三磷酸水解活性的生物化學因子
結論.
肆、 討論
伍、 參考文獻
圖表
附錄
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指導教授 吳惠南(Huey-Nan Wu) 審核日期 2004-1-29
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