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標題: 利用重組Baculoviruses表現流感病毒RNA聚合酵素
Expression of Influenza Viral RNA Polymerase by Recombinant Baculoviruses
作者: 陳筱嬋
Chen, Hsiao-Chan
關鍵字: Recombinant Baculoviruses
出版社: 獸醫公共衛生學研究所
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摘要: A型流感病毒屬於正黏液病毒科(Orthomyxoviridae),其基因體包含八段負股RNA。 每一段流感病毒之基本單位由核蛋白及流感病毒RNA聚合酵素複合體所組合而成(vRNP)。其中,流感病毒RNA聚合酵素複合體由三個次單位 (subunit)蛋白質所構成︰PA、PB1及PB2。於宿主細胞核中,流感病毒之RNA (vRNA) 經由轉錄產生mRNA,也經由複製產生cRNA,cRNA隨即被當成模板,以製造更多的病毒vRNA。在每一段病毒RNA 5’端及3’端之promoter各有13及12個核甘酸為高度保留之序列,可配對形成二級結構,此結構對於promoter之功能行使相當重要。 目前於資料庫中顯示,A型流感病毒株H5N2在病毒(cRNA) promoter區域有部分核甘酸的變異,對於此變異與流感病毒RNA聚合酵素複合體之間的作用是否有影響,為本實驗想要探討之對象。因此本實驗嘗試以baculovirus expression vector system (BEVS)表達出流感病毒RNA聚合酶。BEVS利用一個含有目標基因的transfer vector (pFastBac TM),將此段外源性基因藉由轉位作用(Transposition)嵌入不影響baculovirus複製的基因體區段,重組過後之baculovirus會帶有外源性基因,利用此病毒之polyhedrin (PH) promoter可於感染之昆蟲細胞中表現高量的外源性蛋白。 由於昆蟲細胞可同時被兩種以上攜帶不同表現基因的重組病毒感染;利用此特質,本實驗構築攜帶PA-His、PB1及PB2基因之重組baculovirus, 藉以生產感冒病毒之RNA聚合酵素複合體。 經PCR確認重組病毒之後,於昆蟲細胞中產生病毒子代,並以superinfection的方式產生流感病毒RNA聚合酵素複合體。西方墨點法(Western blot analysis)結果顯示,BEVS可成功表現此三種流感病毒聚合酶次單位。未來將進一步產生流感病毒RNA聚合酵素複合體並利用親合性管柱層析法將之純化,以應用於流感病毒promoter的功能分析。
The genome of influenza A virus is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3' and 5' ends. In virions and infected cells, viral genomic RNAs (vRNA) are tightly associated with the viral NP and P (PB1, PB2, and PA) proteins as ribonucleoprotein (RNP) complexes. Once the RNPs have entered the nucleus of the infected cells, they undergo transcription and replication. The highly conserved noncoding nucleotides in vRNA termini, which form ‘‘panhandle'' or ‘‘fork'' structures by partial complementarity, are important for the transcriptional activity of the viral RNA polymerase. Sequence variations were found in cRNA promoter of HA segment of H5N2 isolates; therefore, the aim of this study was to express influenza polymerase complex for functional analysis of such promoter variants. The recombinant baculovirus expressing individual polymerase was constructed. Western blot analysis confirmed each subunit protein indeed expressed in Sf9 cells infected with recombinant baculovirus. To obtain the polymerase complex, Sf9 cells were super-infected with baculoviruses expressing three individual polymerase subunits that would be purified by means of Ni-NTA affinity chromatography. The structure functional analysis of H5N2 promoter variants will be further conducted by using this recombinant polymerase complex.
其他識別: U0005-2807200913411900
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