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研究生:黃俊評
研究生(外文):Huang Chun-Ping
論文名稱:建立以黃質病毒蛋白質水解酵素為標的之藥物篩選系統
論文名稱(外文):Use of flaviviral proteases as targets to establish drug screening system
指導教授:廖經倫林宜玲林宜玲引用關係
指導教授(外文):Ching-Len LiaoYi-Ling Lin
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:48
中文關鍵詞:黃質病毒蛋白質水解酵素藥物篩選系統
外文關鍵詞:flavivirusproteasedrug screening system
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在本論文中,我們欲建立針對黃質病毒protease為標的之細胞藥物篩選系統,用以篩選出專一性對抗黃質病毒protease的抑制藥物。我們欲藉由偵測報導基因(reporter gene)的表現來反應病毒protease活性,因此設計在黃質病毒非結構蛋白NS2B∕NS3切割處,接上VP16GAL4重組基因,構成一重組受質基因(recombinant substrates gene),使得黃質病毒protease專一性切割重組受質後,導致報導基因能夠表現而被偵測。本實驗所用之報導基因為SEAP(secreted alkaline phosphatase)及luciferase。我們首先利用免疫螢光法來偵測DEN2(dengue virus 2)與JEV(Japanese encephalitis virus)重組受質在BHK-21內的表現情形,發現其可表現於細胞質,且在共同轉染protease基因後,此受質可被切割而進入細胞核中。若再將重組受質、病毒protease及報導基質體共同轉染BHK-21,則可偵測到報導基因之表現,則推測病毒protease可切割重組受質。在轉染不同種之病毒protease的實驗中發現,DEN2 protease可切割本身重組受質,DEN2 protease亦可切割JE 者;而JE protease可切割本身重組受質,但JE protease無法切割DEN2受質。若利用點突變技術(point mutation)將病毒protease三元活化中心之serine置換成alanine,則喪失酵素之活性,在轉染此變種protease後,無法有效切割重組受質,故證明protease的活性對受質的切割非常重要。接著,我們以病毒真正感染上述系統,結果發現無明顯之SEAP活性,顯示自然之病毒protease無法順利切割上述受質。此外,我們在NS2B N端接上Neo基因(Neomycin phosphotransferase gene),以期能加強阻礙未切割之受質進入細胞核的效果;在轉染病毒protease質體的實驗中結果顯示可切割此含Neo重組受質。若以病毒真正感染這Neo重組受質細胞藥物篩選系統後,報導基因活性則有明顯上升。由以上結果顯示,我們已建立一套以黃質病毒protease為標的之細胞藥物篩選系統,並且可利用病毒感染此系統,嚴謹地測試抗病毒藥物之效能,我們冀望由此藥物篩選系統篩選出專一對抗黃質病毒之藥物。

The main purpose of this study is to develop a cell-based high-throughput drug screening system for antiflavivirus agents, specifically aiming at flaviviral proteases as the molecular targets. We have chosen the junction between flavivirus NS2B and NS3 proteins as the specific cleavage site to monitor viral protease activity. To reflect the activities of flaviviral proteases inside the cells, we designed a reporter system by in-frame fusion of VP16-GAL4 hybrid gene with virus nonstructural NS2B gene at 10-a.a. residues downstream of the cleavage junction. Such artificial substrates should be cut specifically by flaviviral proteases, and the resulting VP16-GAL4 hybrid proteins would then enter into the nucleus to turn on the GAL4-drived reporter genes such as SEAP or Luciferase. By immunofluorescent assay (IFA), we observed the cleavage event mediated by flaviviral proteases could change the cellular distribution of artificial substrates from a cytoplasm-mainly to a whole-cell pattern. Using three-plasmid transfection system containing genes of recombinant substrates, viral proteases and reporter, we could readily monitor flaviviral protease activities inside the cells. We found that the engineering DEN2 protease could recognize and cut both DEN2 or JE derived recombinant substrates; in contrast, the JE counterpart could only process its own artificial substrate. Through point mutation technique within catalytic triad, we found the serine residue at 135 of the dengue protease play an important role in its enzymatic activity. Using two-plasmid system containing artificial substrate and reporter genes, we failed to detect activities of natural flavivirus proteases in the infected cells. However, with a further modification by adding Neo gene to the N-terminal of NS2B, we found this new dengue artificial substrate could be properly processed by both the engineered and the natural proteases. Taken together, we have established a cell-based drug screening system that is suitable for not only a flaviviral protease as the single target, but also a flavivirus infection as a whole. We plan to use this technique in automated robot running high-throughput drug screening system for anti-flavivirus agents.

目錄…………………………………………………………………………………. Ⅰ
圖目錄………………………………………………………………………………. Ⅲ
中文摘要……………………………………………………………………………. Ⅳ
英文摘要……………………………………………………………………………. Ⅴ
第一章 前言………………………………………………………………….…….. 1
第二章 材料與方法
一、 細胞株與病毒株……………………………………………………. 6
二、 細胞之培養…………………………………………………………. 6
三、 DNA聚合酵素連鎖反應(polymerase chain reaction, PCR)……… 6
四、 DNA片段之切割、純化、接合及分析……………………………… 7
五、 質體DNA的少量製備(mini-preparation for plasmid DNA)……… 7
六、 細胞轉染 (transfection)…………………………………………… 8
七、 試管內轉錄及轉譯 (in vitro transcription and translation, TnT)… 8
八、 免疫螢光法 (immunofluorescent assay)………………………… 9
九、 Vaccinia virus表現系統與西方墨點法 (western blot)…………… 9
十、 Luciferase Assay……………………………………...……………... 10
十一、 Secreted alkaline phosphatase(SEAP)assay…………………… 10
十二、 重組質體之構築………………………………………………….. 10
第三章 結果
一、 病毒protease NS3質體在細胞內表現NS3蛋白質之情形………….… 15
二、 構築可在細胞內表現的重組受質……………………………………… 15
三、 重組受質、病毒protease與報導基因之三質體篩選系統……………… 17
四、 病毒protease切割重組受質之專一性………………………………..… 17
五、 以三元活化中心點突變法建立喪失酵素活性的登革病毒protease..… 18
六、 以病毒感染測試藥物篩選系統………………………………...……… 19
七、 於DEN2重組受質N端接上Neo基因以加強阻礙未切割進入細胞核的效果…………………………………………………………………… 19
第四章 討論……..…………………………………………………………………. 21
第五章 圖表………………………………………………………………………... 25
參考文獻………………………………………………………………………….… 38
附表……………………………………………………………………………..…... 42
附圖…………………………………………………………………………………. 44
圖一 DEN2 protease重組質體圖示與其在BHK-21之表現……………. 25
圖二 JE protease重組質體圖示與其在BHK-21之表現………….……... 26
圖三 DEN2、JE重組受質基因與報導基因之圖示………………………. 27
圖四 重組受質在細胞內之表現…………………………………………... 28
圖五 重組受質、病毒protease與報導基因之三質體篩選系統…………. 29
圖六 病毒protease切割重組受質之專一性……………………………... 30
圖七 針對三元活化中心以點突變法建立喪失酵素活性的登革病毒protease………………………………………………………………. 31
圖八 利用試管內轉錄與轉譯系統檢測各質體生產突變病毒protease蛋白質的能力…………………………………………………………... 32
圖九 三質體篩選系統偵測突變之登革protease切割DEN2或JE重組受質的能力……………………………………………………………... 33
圖十 登革或JE重組受質及SEAP報導基因雙質體篩選系統測試病毒感染之天然protease活性……………………………………………. 34
圖十一 登革或JE重組受質及Luciferase報導基因之雙質體篩選系統測試病毒感染之天然protease活性………………………………………. 35
圖十二 於DEN2重組受質N端接上Neo基因..…………………………….. 36
圖十三 登革重組受質於加上Neo部分後對病毒感染的效果…………….. 37

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