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研究生:王瑞菁
論文名稱:以pag1基本啟動子取代CMV基本啟動子及增加tet-操作子序列來修飾四環黴素可調控表現系統
論文名稱(外文):The Modification of Tetracycline-Responsive Expression System by Substituting CMV Mini-Promoter for pag1 Mini-Promoter and by Addition of tet-Operator in TRE Region
指導教授:李 松 泰
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
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:2004
畢業學年度:92
語文別:中文
中文關鍵詞:四環黴素反應表現系統昆蟲細胞啟動子
外文關鍵詞:TRESsf-9promoter
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在發展速效性生物殺蟲劑上,轉殖毒性基因之重組桿狀病毒被認為深具潛能。但在發展速效性重組桿狀病毒時,可調控性基因表現系統是一種必要的組件,可避免重組病毒在尚未獲得大量子代病毒時,其毒性基因表現就將細胞毒死,而限制病毒子代產生的數量。四環黴素反應表現系統(Tetracycline-responsive expression system;TRES),由CLONTECH 公司發展出Tet-OnTM及Tet-Off TM兩種可調控性基因表現系統,目前已經成功應用在幾種真核基因表現上。其中Tet-off TM系統被認為適用於發展轉殖毒性基因之重組桿狀病毒。其應用原理是,在田間缺乏四環黴素(Tetracycline;Tc)的情況下,以重組病毒感染昆蟲,即因產生毒性蛋白造成昆蟲快速死亡;反之,在生產製造重組病毒時,加入Tc抑制毒性基因表現,因此可得到高效價之重組病毒。然而, Tet-Off TM系統亦有缺點,主要是在E. coli中有高背景活性,及在昆蟲細胞上調節活化效率不夠想理。在E. coli中的高背景活性,將影響到發展速效性重組病毒殺蟲劑的毒性基因選殖;而在昆蟲細胞上調節活化效率亦不夠好,更將影響重組病毒殺蟲劑的殺蟲效果。本試驗藉由Hz-1病毒pag1基本啟動子取代Tet-Off TM系統中的CMV基本啟動子,及增加Tet-Off TM系統中反應質體上四環黴素抑制子結合的操作子(tet-O)序列,來改進Tet-Off TM基因表現系統,使其更適合於速效性重組桿狀病毒殺蟲劑的開發利用。
本論文研究結果發現,未經修飾之反應質體pTRE-Luc,在E. coli中其背景活性表現量高;在昆蟲細胞上受tTA調節時,其活化倍率達300倍左右。以乳糖操作子(lac operator)修飾之反應質體pTRE-op-Luc及pTRE-opr-Luc,可降低在E. coli背景活性約27~50%。若短暫性轉染反應質體至昆蟲細胞,在活化子tTA存在時,pTRE-opr-Luc表現活性不僅較pTRE-Luc高,其活化倍率亦可提高到691倍。pTRE-op-Luc受到活化子tTA活化時的活性雖較pTRE-Luc低,但其活化倍率亦有350倍。另外,以Hz-1病毒pag1基本啓動子取代CMV基本啓動子之反應質體pTRE-69 Luc及pTRE-90Luc,可更明顯降低啓動子的在E. coli中背景活性。而在昆蟲細胞的調節表現時,以pag-90基本啓動子構築的反應質體(pTRE-90Luc),其活化表現量是未經修飾之pTRE-Luc的2倍,但在昆蟲細胞上其背景活性亦相對的提高。因此,當tTA存在且不含Tc的情況下,只得到61倍的活化倍率。pTRE-69 Luc的活性表現較pTRE-90Luc低,但也比pTRE-Luc高。而它在昆蟲細胞上的背景活性較pTRE-90Luc低,因此整體的活化倍率反而比pTRE-90Luc高,達到153倍。在所有的反應質體中又增加四環黴素反應區域(Tetracycline-responsive element;TRE,即七個tet-O序列),在昆蟲細胞內可以提高活化表現倍率及降低背景活性。增加七個tet-O序列的pTRE-90Luc/TRE,受tTA的活化可提高247倍的活性。而pTRE-90Luc/2TRE是增加14個tet-O序列,則可增加到1750倍。本試驗證實經修飾的TRES,可有效提升可調控性的基因表現系統在昆蟲細胞的表現效率,對於速效性重組桿狀病毒殺蟲劑的發展,將具有具體的可實用性。

Engineered recombinant baculoviruses harboring toxin genes have increased the potential for the development of fast-acting recombinant viral insecticides. The availability of regulatory gene expression systems which allow the tight and specific regulation of toxin gene expression are required for the development of engineered biopesticides, since the expression of a toxin genes during propagation of the recombinant virus will limit the mass production of virus. The tetracycline-responsive expression system (TRES) has been successfully employed in several eukaryotic gene expression systems. Of the two types of TRES, Tet-OnTM and Tet-Off TM, the Tet-Off TM is suitable for the development of engineered baculoviruses harboring toxin gene. In the absence of tetracycline(Tc), a recombinant virus containing a toxin gene will express the toxin protein in the infected insect cells. Therefore, the insects in the fields would be killed. In contrast, when grown in tissue culture in the presence of Tc, it should be possible to propagate the recombinant virus to very high titer because the expression of the toxin gene will be turned off. However, the Tet-off has some disadvantages since high background expression in E. coli and low activation efficiency in insect cells. The high background expression in E. coli will limit the successful cloning of the toxin gene and the low activation efficiency will hinder the fast acting of recombinant viral pesticide. To circumvent these difficulties, we modified the Tet-Off TM system by substituting CMV mini-promoter for pag1 mini-promoter and addition of one more tet-operators in TRE region of response plasmids.
In this study we found that unmodified response plasmid pTRE-Luc had high background expression in E. coli and had 300-fold activation by tTA in insect cell. We also evaluated two modified response plasmids with addition of lac operator to TRE region. Results showed that 27-50 % reduction of background expression in E. coli were observed with two lac operator modified response plasmids(pTRE-op-Luc and pTRE-opr-Luc). A 691-fold increase of promoter activity was detected upon activation by tTA in insect cells transfected with one of the lac operator modified response plasmids pTRE-opr-Luc. pTRE-op-Luc showed a 350-fold activation by tTA and low background expression level comparing with pTRE-Luc. In the other modified response plasmids(pTRE-69 Luc and pTRE-90Luc), CMV minimal promoter were replaced by the pag-1 gene minimal promoter of Hz-1 virus. Results showed that significant reductions in promoter activity were observed in E. coli with these modified response plasmids. In insect cell, the luciferase activity of modified response plasmid pTRE-90Luc was 2-fold increase comparing with unmodified response plasmid pTRE-Luc. However, the basal level of response plasmids with pag-90 minimal-promoter also increased in insect cell. The pTRE-90Luc showed a 61-fold increase of promoter activity upon activation by tTA in the absence of Tc. pTRE-69Luc showed a 153-fold activation by tTA and were low background expression comparing with pTRE-90Luc in insect cell. Addition of tet-operators in TRE region of all the response plasmids, the activation efficiency increased significantly and the basal level activity decreased in insect cells. The pTRE-90Luc/TRE, which has 7 extra tet-operators, showed a 247-fold increase of promoter activity upon activation by tTA, while the pTRE-90Luc/2TRE, which has 14 extra tet-operators, showed a 1750-fold increase of promoter activity upon activation by tTA.
These results suggest that these modified TRES will be useful for the development of fast-acting recombinant viral insecticides.

中文摘要 .....................................................i
英文摘要 ...................................................iii
誌 謝 ....................................................v
目 次 ...................................................vi
圖 目 錄 .................................................viii
第 一 章 前言 ................................................1
第 二 章 文獻探討.............................................4
2.1 桿狀病毒.................................................4
2.1.1桿狀病毒之分類..........................................4
2.1.2 桿狀病毒之構造.........................................5
2.1.3 桿狀病毒之生活史.......................................5
2.1.4 桿狀病毒之基因表現.....................................5
2.2 桿狀病毒之應用...........................................6
2.2.1 生物性農藥.............................................6
2.2.2 基因療法...............................................7
2.2.3 外源蛋白表現系統.......................................8
2.3 可誘導基因表現系統.......................................8
2.3.1 在哺乳動物細胞之應用...................................9
2.3.2 在昆蟲細胞之應用......................................10
2.3.3 tet-off/tet-on系統之改進策略..........................10
第 三 章 材料與方法..........................................12
3.1 試驗材料................................................12
3.2 試驗方法................................................15
第 四 章 結果 ...............................................19
4.1 以Hz-1桿狀病毒pag1啟動子構築之反應質體的活性探討........19
4.1.1 在大腸桿菌(E. coli)中的活性.....................19
4.1.2 在昆蟲細胞(Sf-9)中之活性............................20
4.1.2.1 測試Tet-Off TM系統的最佳組合........................20
4.1.2.2 修飾的反應質體在Sf-9細胞之活性探討..................21
4.2 增加TRE片段對反應質體活性的探討.........................22
4.2.1 反應質體構築......................................22
4.2.2 增加tet-O序列之反應質體在E. coli的背景表..........23
4.2.3 增加tet-O序列之反應質體在Sf-9細胞表現活性.........23
4.2.4 TRE中tet-O數目對反應質體活性之影響................24
第 五 章 討論................................................25
第 六 章 總結................................................29
參考文獻.....................................................42
附 錄.....................................................46

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