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標題: | 感染性胰臟壞死病毒之鳥嘌呤核?轉移?及病毒基因體連接蛋白特性之研究 Characterization of Guanylyltransferase and Genome-Linked Viral Proteins in Infectious Pancreatic Neorosis Virus (IPNV) |
作者: | 劉銘燦 |
出版年 : | 1992 |
學位: | 碩士 |
摘要: | 感染性胰臟壞死病毒之病毒蛋白質VPI目前除了可能具RNA聚合?功能外,本實驗證明VPI具鳥嘌呤核?轉移?的特性。以[α-32P]GTP同病毒反應,且以鎂離子為輔助因數,反應20分鐘後,直接以SDS-PAGE分析,可偵測到VPI與[α-32P]GTP結合的複合體,故認為VPI有鳥嘌呤核?轉移?的功能。此酵素對GTP的結合有專一性。此種GMP-酵素複合體的形成會受焦磷酸(pyrophosphate, PPi), DTT, EDTA, NaCl抑制;當加入活體外轉錄(in vitro transcription)的RNA時,GMP-VPI複合體消失。當改變反應溫度及pH值時會影響鳥嘌呤核?轉移?的活性,且會使VPI降解為小分子的蛋白質。除此之外,在本篇報告中我們也將討論感染性胰臟壞死病毒之病毒基因體連接蛋白(VPg)的性質,此蛋白質具有與[α-32P]GTP及[α-32P]UTP結合的能力,以RNase A處理以[α-32P]GTP及[α-32P]UTP標定之VPg-RNA,會產生57kDa及33kDa二種蛋白質。感染性胰臟壞死病毒之鳥嘌呤核?轉移?及病毒基因體連接蛋白,可能與病毒的轉譯作用及病毒RNA合成有關。 The putative RNA polymerase, VP1, in infectious pancreatic necrosis virus (IPNV) was shown to form an enzyme-guanylate intermediate which is indicative of guanylyltransferase activity. The activity associated with VP1 was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virion was incubated with [α-32P]GTP. The reaction is specific for GTP and requires divalent cations as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferase. The formation of GMP-enzyme complex was inhibited by pyrophosphate (PPi), DTT, EIDTA, NaCl, and in vitro transcriptional RNA. VP1 is sensitive to temperature, and the pH which make VP1 degrade to small peptides. In addition to VP1, we also demonstrated that IPNV has the genome-linked viral protein (VPg) which can associate with [α-32P]GTP and [α-32P]UTP. Ribonuclease treatment of [α-32P]GTP or [α-32PJUTP labelled VPg-RNA released 57 kDa and 33 kDa two polypeptides. VPg and Guanylyltransferase of IPNV may be involved in translation and viral RNA synthesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75860 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
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