Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/14181
標題: 利用果蠅細胞表現牛傳染性鼻氣管炎病毒去氧尿核甘三磷酸鹽水解酵素
Expression of infectious bovine rhinotracheitis virus dUTPase in Drosophila schneider 2 cell
作者: 王中聖
關鍵字: 牛傳染性鼻氣管炎病毒
IBRV
去氧尿核甘三磷酸鹽水解酵素
果蠅細胞
dUTPase
S 2 cell
出版社: 獸醫學系
摘要: 此研究是將本實驗室先前已選殖出並定序的臺灣分離雲林(YL)株牛傳染性鼻氣管炎病毒(infectious bovine rhinotracheitis virus;IBRV)去氧尿核甘三磷酸鹽水解酵素(dUTPase)基因的完整轉譯序列(coding sequence)嵌入果蠅表現載體 (expression vector)pAc5.1/V5-His C,置於Ac5.1啟動子(promoter)的下游,構築成重組質體pAc5.1/HJ4。隨後將該重組質體對果蠅細胞(Drosophila Schneider 2 cells;S2 cells)進行短暫性轉染(transient transfection),使細胞產製分子量約40 kDa的IBRV dUTPase融合型蛋白(fusion protein)。萃取表現此融合蛋白的細胞液,使用瓊脂膠體電泳(SDS-polyacrylamide gel electrophoresis;SDS-PAGE)進行電泳分析,雖無法在重組基因產物預期的大小位置發現環帶(band),但將細胞蛋白萃取物通過嵌有鎳離子的親和性層析管柱(nickel-chelating affinity chromatography column),純化出氨基端帶有六個組氨酸標幟(6X histidine tag)的目標蛋白,隨後以西方墨點法(Western blot)偵測此目標蛋白,結果可在分子量大小約為40 kDa的預期位置呈現單一環帶(band)。此實驗結果顯示所建立IBRV dUTPase基因在果蠅細胞內表現與其融合型蛋白質的純化系統可提供分析dUTP水解酵素在IBRV及真核細胞內其蛋白分子特性差異的研究並且提供抗病毒化學藥物的設計與研發的參考。
Abstract The gene that codes for the dUTPase of infectious bovine rhinotracheitis virus (IBRV) YL strain was attempted to place downstream on the promoter (Ac5.1) of the expression vector (Ac5.1/V5-His C) for constructing a recombinant expression plasmid (pAc5.1/HJ4) in the Drosophila Schneider 2 cells (S2 cell). After transfection, the IBRV's dUTPase was expected to expression a recombinant protein with ~40 kDa in the S2 cells. Unexpectedly, examination of SDS-PAGE analysis with cellular proteins in the transfected S2 cells did not appear a visible band for the expressed protein. Since there are six histidine residues at the N-terminal end of the recombinant protein, a nickel-chelating affinity column chromatography could be used for purification of the IBRV's dUTPase. In addition, the immunoblotting against T-7 tag monoclonal antibody was applied to enhance the sensitivity for detecting the expressed protein in the S2 cells. The results obtained with protein purification by affinity chromatography and detection by western blot indicated that the recombinant protein existed in the transfected S2 cells. Thus, the IBRV's gene encoding dUTPase could be constructed, transfected, and expressed in the S2 cells. This study provided a useful information on biochemical characterization of the viral protein expressed in the eukaryotic host cells, which might be important for pharmaceutical interests of antiviral drug design.
URI: http://hdl.handle.net/11455/14181
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