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題 名 | High-Level Expression of the Infectious Bovine Rhinotracheitis Virus dUTPase in Escherichia Coli=牛傳染性鼻氣管炎病毒去氧尿核甘三磷酸鹽水解酵素在大腸桿菌之高量表現 |
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作 者 | 鍾楊聰; 王斐; | 書刊名 | 臺灣畜牧獸醫學會會報 |
卷 期 | 65:4 1995.12[民84.12] |
頁 次 | 頁327-337 |
分類號 | 437.246 |
關鍵詞 | 牛傳染性鼻氣管炎病毒; 去氧尿核甘三磷酸鹽水解酵素; 基因表現; Infectious bovine rhinotracheitis virus; dUTPase; Gene expression; |
語 文 | 英文(English) |
中文摘要 | 去氧尿核甘三磷酸鹽水解酵素(dUTPase)普遍存在於自然界,在細胞之核甘酸代謝過程中扮演著重要的角色。此酵素可催化dUTP使轉變成dump和焦磷酸鹽(pyrophosphate),除了阻止Dutp被誤嵌入DNA分子之外,還且具有提供Dump使最後轉換成Dttp的功能。參考國外已發表的研究報告,我們已經自行分離並鑑別出台灣YL株牛傳染性鼻氣管炎病毒製造dUPTase的基因,並將該基因的轉譯序列(coding sequence)嵌入表現載體Pet-28a(+)中,置於噬菌體T7基因10啟動子(promoter)的下游。我自接著將此重組質體送入大腸桿菌BL21 (DE3) pLysS細胞使轉形,並適時以異丙基硫化半乳糖甘(IPTG)刺激細胞中T7 RNA聚合酵素的合成,期使其能強力驅使T7啟動子經由轉錄與轉譯作用合成dUPTase酵素。使用SDS-PAGE分析經IPTG激化過之細胞的萃取蛋白,發現在分子量相當於37 kDa處出現一非常明顯的環帶(band),和預期的重組基因產物的大小相符;而未受激化的對照組則闕如。此外此環帶的強度伴隨著加入IPTG時間的長度而增強,粗估其產量最高可達全部蛋白質量的30%。這些結果顯示本篇報告所描述的表現系統可製造出大量的IBRVdUTPase,俟將來進一步純化和測定其活性後,可用來研究酵素結構的與功能間之關係。 |
英文摘要 | Deoxyuridine 5'-triphosphte necleotidohydrolase (dUTPase), widespread in nature with a crucial function in the nucleotide metabolism, catalyzes the hydrolysis ofdUTP to dUTP and function in the mcleotide metabolism, catalyzes the hydrolysis of function relationship of the enzyme we have cloned and identified the gene encoding the dUTPase of infectious bovine rhinotracheitis virus (IBRV) strain YL previously isolated in Taiwan and expressed the protein in the Escherichia coli system. The coding region of this gene starting with codon 22was positioned downstream of the phage T7 gene 10rpomoter on the expression vector Pet28a(+). Transformation of this recombinant into E. coli BL21(DE3) pLysS cells containing the T7 RNA polymerase, under the control of the lacUV5 promoter, allowed expression of the protein on induction of the T7 RNA polymerase by isopropyl β-D-thiogalactopyranoside(IPTG.). SDS-polyacrylamide gel electrophoresis analysis of cellular proteins from IPTG-induced cells revealed the presence of a prominently stained band corresponding to a molecular mass of 37 kDa, which is in good agreement with the prediction from the DNA sequence. It was also observed that the intensity of the 37 kDa band increased with time of incubation with IPTG. Densitometric comparisons with stained protein standards indicated that the recombinant protein constituted about 30% of the total cellular protein. Taken together, these results suggest that the overproducing system described here is suitable for providing sufficient amounts of IBRV dUTPase for furtheir investigations. |
本系統中英文摘要資訊取自各篇刊載內容。