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研究生:楊豐名
研究生(外文):Feng-Mine Yang
論文名稱:探討IRES序列於開發新的桿狀病毒表現系統
論文名稱(外文):The use of IRES elements for novel baculovirus expression system
指導教授:吳宗遠
指導教授(外文):Tzong-Yuan Wu
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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:75
中文關鍵詞:間接核醣體認知序列桿狀病毒
外文關鍵詞:IRESBaculovirus
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桿狀病毒 (Baculovirus) 為雙股環狀DNA病毒,容易利用基因工程做基因修飾,藉著多角體基因強起動子的調控,可以產生大量的異源蛋白,為一廣為使用的蛋白表現系統。本實驗有四個主要目標:(I)利用encephalomyocarditis virus(EMCV)的IRES,來發展桿狀病毒表現系統的 bi-cistronic 表現系統。(II)分析比較三種不同IRES(EMCV、HCV和EV71)在昆蟲細胞中的活性表現,希望能在昆蟲細胞中找到具有高度活性表現的IRES序列。(III)利用EMCV-IRES序列及水母(Aequorea victoria)的綠螢光蛋白基因(green fluorescent protein, GFP)發展易篩選的重組桿狀病毒轉殖載體系統。(IV)利用IRES來建立篩選藥物的模式,以便利用此系統來發展抗病毒藥物快速篩選的工具。
Bi-cistronic的重組病毒之構築,是將EMCV-IRES放入含有polyhedrin啟動子的載體pBlueBac 4.5中,接著把珊瑚的紅螢光DsRed基因及水母的綠螢光GFP基因分別放在IRES序列的前後,再與病毒DNA重組,得到bi-cistronic重組病毒。藉由被感染的昆蟲細胞同時發出的紅螢光和綠螢光,我們證明EMCV-IRES序列在昆蟲細胞仍有活性。
為了進一步確定不同病毒的IRES是否也能在昆蟲細胞中表現,分別利用β-gal和SEAP做為報告基因(report gene),接到同時含有CMV和p10起動子的pTriEx-4載體上。再把EV71、HCV和EMCV病毒的IRES接到β-gal和SEAP兩個基因中間。利用IRES起動SEAP基因,藉由分析SEAP基因的表現,以檢測IRES的活性。結果顯示,EV71-IRES序列在昆蟲細胞表現系統中效果最好。同時, bi-cistronic 轉殖載體提供了一個快速、簡單挑選重組病毒的方法,我們以SEAP基因以及GFP基因分別置於EMCV-IRES序列之前後,可藉由被感染細胞發出的綠螢光,很輕易的篩選出重組成功的目標病毒,這對桿狀病毒表現系統在使用上將更為便利。
IRES對RNA病毒來說是一段不可缺少的序列,如果能破壞或拮抗IRES序列對核醣體的結合,就能阻斷病毒的繁殖。先前的研究顯示,藥物IFN-α可能與抑制HCV-IRES序列的活性有關,另一方面,也有研究指出IFN-α無法抑制HCV-IRES序列的活性,因此嘗試以IFN-α為測試藥物分析其在Huh7細胞中對EV71-IRES和HCV-IRES活性的影響,我們的結果顯示IFN-α對HCV-IRES及EV71-IRES序列皆有顯著的活性抑制,因此我們可藉由bi-cistronic載體上報導基因表現的強弱建立一方便快速找尋針對拮抗病毒IRES序列的藥物篩選系統。

Baculovirus is a double strain DNA virus, easily to make gene modification by gene engineering that use strong promoter to produced large heterogametic protein is wildly applied in the production of numerous recombinant protein. The aims of this study were to (I) used EMCV-IRES to development bicistronic vector of baculovirus expression system, (II) activity analyze of three different IRES from EMCV、HCV and EV71, to search which IRES has highly activity in insect cells, (III) used EMCV-IRES and green fluorescent protein to development recombinant virus screening, (IV) used IRES to establish a rapidly screening anti-virus compounds model.
Construction of the recombinant bicistronic virus was achieved by transfer an IRES element from EMCV into transfer vector pBlueBac4.5 under the control of a strong polyhedrin promoter. The report gene green fluorescent protein (GFP) and (DsRed), constitute the first and second cistrons, respectively, of the same transcript. The transfer vector and virus DNA were cotransfected into Spodoptera frugiperda (Sf21) cells. The recombinant virus was observed by detecting the emission of red and green fluorescence from the infect cells. However, we proved the IRES element. Our result demonstrate the EMCV-IRES element does support effective translation in insect cells.
In order to sure different IRES element does also support effective translation in insect cells. Construction of the recombinant bicistronic virus was achieved by transfer the report gene of β-gal and SEAP into transfer vector pTriEx4 under the control of a strong CMV and p10 promoter. And then constructs the EMCV-IRES、HCV-IRES and EV71-IRES element was cloned in between the two report gene. The IRES element enabled efficient translation of the second cistron as determined by SEAP activity in infected cells. The result indicated EV71-IRES element has highly effective translation in insect cells. At the same time, bicistronic transfer vector provide a simple way to isolate recombinant virus. The recombinant virus was isolated by detecting the emission of green fluorescence from infected cells.
The lack of IRES element was not due to RNA virus propagation. Preview study, IFN-α could be inhibit the IRES’s activity from HCV. On the other hand, some research indicated IFN-α can not inhibit the IRES’s activity from HCV. So we attempted to analyze IFN-α as test medicine influence with IRES from HCV and EV71 in Huh-7 cells. Our result demon ate IFN-α has highly inhibit on HCV-IRES and EV71-IRES. So we can use bicistronic vector to build a rapidly screening compound system.

英文摘要................................................1
中文摘要................................................3
緒論....................................................5
材料與方法.............................................13
壹、重組桿狀病毒bi-cistronic表現系統之建立
一、 細胞與病毒..................................13
二、 質體........................................13
三、 質體轉型....................................14
四、 質體的純化..................................15
五、 質體轉染....................................17
六、 終點稀釋法..................................17
七、 聚合酶連鎖反應..............................18
八、 Secreted Human Placental Alkaline Phosphatase活性檢測
............................................18
九、 西方點墨法試劑、緩衝溶液與抗體..............19
貳、不同IRES之活性檢測
一、 細胞與病毒..................................22
二、 質體........................................23
三、 質體轉型....................................24
四、 質體的純化..................................24
五、 質體轉染....................................24
六、 終點稀釋法..................................25
七、 聚合酶連鎖反應..............................25
八、 Secreted Human Placental Alkaline Phosphatase活性檢測
.....................................................26
九、 β-galactosidase活性檢測.....................27
十、 藥物試驗....................................28
結果.................................................29
壹、重組桿狀病毒bi-cistronic表現系統之建立
一、 Bi-cistronic質體的構築............ .........29
二、 短暫性細胞轉染分析實驗......................30
三、 重組病毒的構築與純化........................31
四、 昆蟲細胞的感染..............................31
五、 在昆蟲細胞中,比較腦脊髓炎病毒(EMCV)IRES的活性...................................................32
六、 桿狀病毒偵測標誌的建立......................34
貳、不同IRES之活性檢測
一、 IRES序列的取得..............................35
二、 構築一個含有兩種報告基因(report gene)的bi-cistronic載體.................................................37
三、 在哺乳類細胞中,不同IRES活性檢測之短暫性細胞轉染分析...................................................39
四、 重組病毒的構築與純化........................40
五、 昆蟲細胞的感染,檢測不同病毒IRES活性之分析..40
六、 藥物分析試驗................................41
討論.................................................43
圖表.................................................51
參考文獻.............................................71
附錄
一、 pBlueBac4.5 plasmid 圖
二、 pTriEx-4 plasmid圖
三、 pEGFP-C1 plasmid圖
四、 pIRES-EGFP plasmid圖
五、 pDsRed1-N1 plasmid圖
六、 pSEAP-control plasmid圖
七、 pβ-gal-control plasmid圖

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