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研究生:陳素良
研究生(外文):Chen, Su-Liang
論文名稱:利用基因工程建立目宿夜蛾核多角體病毒之偵測及其控制性表現系統
論文名稱(外文):The establishment of detection & control gene expression systems AcMNPV by genetic engineering
指導教授:趙裕展
指導教授(外文):Chao Yu-Chan
學位類別:碩士
校院名稱:中國文化大學
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:1997
畢業學年度:85
語文別:中文
論文頁數:4
中文關鍵詞:目宿夜蛾核多角體病毒控制性表現系統病毒偵測
外文關鍵詞:AcMNPVexpression systemviral detection
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為了使桿狀病毒更趨於良好的生物性殺蟲劑,本實驗有兩個主要的目標:
(i)利用水母的綠螢光基因(green fluorescent protein, GFP),來完成
病毒的偵測工作。(ii) 找到四環黴素調控系統(tetracycline-
controlled gene expression system),在昆蟲細胞中最適合的調控條件
,以便利用此系統,來完成害蟲防治的工作。含有GFP基因的重組病毒之
構築,是將水母(Aequorea victoria)的GFP基因放入含有polyhedrin基因
的載體pAcUW21中,再與病毒DNA發生重組,GFP基因是由p10起動子所起動
的。在純化病毒時,可藉由被感染的細胞發出的綠螢光,而輕易的得到重
組病毒。在昆蟲細胞中使用四環黴素調控系統時,短暫性細胞轉染分析之
DNA濃度為1 ug/2×105 cell,而之後病毒感染moi在0.1時,轉染的效率
會最好。另外,四環黴素 (tetracycline, Tc) 濃度只要1 ug/ml就可以
將目標基因的表現量壓抑下來。活化性重組病毒vAcP10tTA的構築,是將
含有Tc repressor及VP16活化區的融合基因 (tTA),接到pAcUW21載體上
, 再與病毒DNA發生重組,tTA同樣是由p10起動子起動。總共挑到了十株
重組病毒,為了選出活化效果最好的病毒,故在三種細胞株內測其活性
,(Sf9, Sf21 and TN368),感染72小時後,10株重組病毒的活性,在Sf9
細胞株中以vAcP10tTA-1最好;在Sf21細胞株中以vAcP10tTA-5最好;而在
Tn368細胞株中則以vAcP10tTA-4最好。

The aims of this study were to (i) produce a green
fluorescent protien (GFP) recombinant virus for detection of
viral infection, (ii) optimize the condition of tetracycline-
controlled gene expression system in insect cells.
Construction of the recombinant GFP virus was achieved by
transferring a GFP gene from jellyfish (Aequorea victoria) into
a polyhedrin gene-containing transfer vector pAcUW21 under the
control of a strong p10 promoter of Autographa californica
multiple nuclear polyhedrosis virus (AcMNPV). The transfer
vector and AcMNPV DNA were cotransfected into Spodoptera
frugiperda (Sf21) cells. The recombinant virus was isolated by
detecting the emission of green fluorescence from the infected
cells. The optimal conditions used for tetracycline-
controlled gene expression were 1 ug pTRE-LUC /2×105 cells at
viral m.o.i. 0.1. Expression of luciferease activity was
inhibited when tetracycline (Tc) was added to the mixtures at 1
ug Tc/ml.Contruction of an active-recombinant virus, vAcP10tTA,
was completed by transferring a fusion gene (tTA) containing a
tetracycline repressor and a VP16 transcription active domain
into a transfer vector pAcUW21. After identification, 10
recombinant viral colonies were selected. To obtain the best
active-recombinant virus, three cell lines (TN368, Sf9 and Sf21)
were used and transfected with pTRE-LUC. After 24 h of
transfection, the cells were infected with the 10 vAcP10tTAs,
respectively. Luciferase activity was assayed from each
infectant after 72 h of infection. The results showed that a
highest luciferase activity expressed in Sf9 was vAcP10tTA-1, in
Sf21 was vAcP10tTA-5 and in TN368 was vAcP10tTA-4.

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