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題 名 | 重組型牛傳染性鼻氣管炎病毒DNA-尿密定醣甘酵素之產製與純化=Production and Purification of Recombinant Infectious Bovine Rhinotracheitis Virus Uracil-DNA Glycosylase |
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作 者 | 鍾楊聰; 徐維莉; 劉新梧; | 書刊名 | 臺灣畜牧獸醫學會會報 |
卷 期 | 66:4 1996.12[民85.12] |
頁 次 | 頁265-275 |
分類號 | 437.246 |
關鍵詞 | 牛傳染性鼻氣管炎病毒; DNA-尿密定醣甘酵素; 融合型蛋白; 蛋白質純化; Infectious bovine rhinotracheitis virus; Uracil-DNA glycosylase; Fusion protein; Protein purification; |
語 文 | 中文(Chinese) |
中文摘要 | DNA-尿密定醣甘酵素(uracil-DNA glycosylase)簡稱UDGase,是細胞執行DNA修 補作用(DNA repair)的重要酵素之一。此酵素廣泛存在於自然界各種生物體,包括皰疹病毒 (herpesvirus)。牛傳染性鼻氣管炎病毒(infectious bovine rhinotracheitis virus; IBRV) 隸屬於Alphaherpesvirinae病毒亞科,其基因體(genome)之單長區(unique long region) 核甘酸序列分列分析顯示此區段包含有一長615個核甘酸的開讀框(open reading frame)UL2,其所預測之蛋白質產物包括一段UDGase的保留性標識序列(signature sequence)。為了獲得足量之IBRV UL2蛋白進行其生化特性之相關研究,我們自本實驗室先 前已製備得之IBRV(台灣YL株)病毒基因體DNA(genomic DNA)中選殖出含有UL2開讀框的DNA 片段,並分別將之轉接於原核表現載體(prokaryotic expression vector) pET-28b(+)與 pET-32b(+)中,然後送入大腸桿菌(Escherichia coli) BL21(DE3)[pLys S]細胞中轉形。在 異丙基硫化半乳糖甘(IPTG)的刺激下,這些細胞可生產出與預期相符的分子量約為21 kDa 與38 kDa的融合型蛋白(fusion protein)。利用金屬離子親和性層析法(metal affinity chromatography)可快速將該等產物純化為均質。純化後的融合蛋白經功能測試顯示皆具有 UDGase活性,但由pET-28b(+)載體衍生所得的融合蛋白的酵素活性較另者強約十倍。 |
英文摘要 | Uraci-DNA glycosylase (UDGase) is an essential component of the cellular DNA repair machinery. It is present in a wide range of organisms, including herpesviruses. Infectious bovine rhinotracheitis virus (IBRV), an important pathogen of cattle, is a member of the subfamily Alphaherpesvirinae. Nucleotide sequence analysis within the unique long segment of IBRV genome identified an open reading frame (ORF) designated UL2 whose deduced protein product of 204 amino acids contained a consensus UDGase signature sequence. To obtain sufficient amounts of IBVR UL2 protein for biochemical characterization, the DNA segment containing the UL2 sequence was cloned and separately inserted into the prokaryotic expression vectors pET-28b(+) followed by transformation into Escherichia coli BL21(DE3)[pLysS] sells. Upon induction with isopropyl-β-D-thiogalactopyranoside these cells produced a 21-kDa and 38-kDa fusion protein, respectively. A column chromatographic procedure using metal affinity resins resulted in homogenous preparations of these proteins. Both of the purified fusion proteins exhibited specific UDGase activities in the in vitro enzyme assay, but the detectable activity of the protein derived from pET-28(+) was tenfold higher than that of another. |
本系統中英文摘要資訊取自各篇刊載內容。