研究生: |
陳詩文 Chen, Shih-Wen |
---|---|
論文名稱: |
利用昆蟲桿狀病毒強起動子P(-90)大量表現外源基因 The establishment of a foreign gene expression system using |
指導教授: |
李銘亮
Li, Ming-Liang 趙裕展 Chao, Yu-Chan |
學位類別: |
碩士 Master |
系所名稱: |
生命科學系 Department of Life Science |
畢業學年度: | 84 |
語文別: | 中文 |
論文頁數: | 2 |
中文關鍵詞: | 桿狀病毒 |
英文關鍵詞: | AcMNPV, Hz-1 |
論文種類: | 學術論文 |
相關次數: | 點閱:195 下載:0 |
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在研究HzV-1桿狀病毒早期基因表現時, 本實驗室發現一個極早期基
因 pag1 具有極強的起動子活性, 而做起動子序列刪除(
promoterdeletion)分析時, 發覺當保留從轉錄起始點(+1)為基點往
上(-90 bp)及往下(+29bp)的區域, 簡稱P(-90), 具最強的起動子活性.
而於鱗翅目細胞株SF9, SF21及果蠅細胞株SL2做短暫表現(
transcienttransfection assay)分析時, 也發現其較常用的起動子具較
佳的起動子活性. 而本實驗則證實以P(-90)來表現外源基因的重組病毒仍
具極強起動子活性且較多角體起動子為早20-16小時 ;而lacZ活性分析與
西方點墨分析結果顯示其有近於多角體基因起動子活性.另以gfp(green
fluorescent protein)為報導基因時,測得其表現量可達P10起動子表現量
的三分之 一, 故認為其具有發展為新置換載體之潛能.並構築了以之為起
動子的置換載體,命名為pHgP(-90),期能用之表現外源基因並得高產量且
具較佳活性之外源蛋白.
The very early gene pag1 was found to express high level of
promoter activity during the investigation of the expression of
the early genes of baculovirus HzV-1. The level of its promoter
activity was found highest from the upstream -90 to the
downstream +29 (the transcription start site as +1), described
as p(-90), by the subsequent deletion experiments.In addition,
the activity of p(-90) was found to be higher than most
promoters expressed in the Lepidopteran cell lines such as SF9,
SF21 and the Drosophila cell lines SL2 in the transcient assays
(Lee, 1995). The aim of any project is to investigate whether
the activity of P(-90)expressed in the recombinantAutographa
californica nuclear polyhedrosis virus using lacZas the repoter
gene is as hihh as that in transcient transfection assay. If it
is, a transplacement vector with P(-90) as the promoter will be
construsted to express foreign genes with higher yields ans
activities. The results of this project show that although the
levels of the B-galactosidase measured by the western blotting
experiments and the activity assays are similar those driven by
polyhedrin gene promoter, the expression of lacZ driven by
promoter P(-90) is 18 hours earlier than that driven by the
polyhedrin gene promoter in the recombinant AcMNPV.
The very early gene pag1 was found to express high level of