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題 名 | Purification and Functional Analysis of the Infectious Bovine Rhinotracheitis Virus dUTPase Expressed in Escherichia Coli=重組型牛傳染性鼻氣管炎病毒去氧尿核甘三磷酸鹽水解酵素之純化與功能分析 |
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作 者 | 鍾楊聰; | 書刊名 | 臺灣畜牧獸醫學會會報 |
卷 期 | 66:2 1996.06[民85.06] |
頁 次 | 頁71-82 |
分類號 | 437.257 |
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; 重組蛋白; 鎳離子親和性層析法; Infectious bovine rhinotracheitis virus; dUTPase; Recombinant protein; Nickel-chelating affinity chromatography; |
語 文 | 英文(English) |
中文摘要 | 去氧尿核廿三磷酸鹽水解酵素(deOXyuridine 5'-triphosphate nucleoTIdohydrolase, dUTPase)可催化使dUTP轉變成dUMP與焦磷酸鹽之反應,在細胞之核 甘酸代謝與DNA複製過程中扮演著重要的角色。本實驗室先前曾選殖並定序台灣分離YL株牛 傳染性鼻氣管炎病毒(infectionus bovine rhinotracheitis virus,IBW)製造dUTPase的基 因。在本研究中,我則將該基因完整的轉譯序列(coding sequence)嵌入表現載體( expression vector)pET28b(+)中,置於噬菌體T7啟動子(promoter)的下游,構築成重組質 體pET/HJ4。隨後將該質體送入大腸桿菌BL21(DE3)[pLysS]細胞中,經異丙基硫化半乳糖甘 (IPTG)的激化可促使細胞產製重組蛋白(recombinant protein)lBRV dUTPase,其含量約佔 全部可溶性蛋白的1-2%。將細胞之可溶性蛋白萃取物通過嵌有鎳離子的親和性層析管柱 (nickel-chelating affinity chromatographic column),可快速純化到胺基端帶有源自 表現載體之六個組胺酸標記(histidine tag)的重組型dUTPase。此純化所得之產物在SDS- PAGE分析中呈現分子量大小約為4OkDa的單一環帶,並在功能測試中展現使dUTP轉變為 dUMP的催化能力(每毫克蛋白質在每分鐘內可使41μmol之受質水解)。這些實驗結果顯示 我所建立的基因表現與蛋白質純化系統可提供足量的具有酵素活性的IBRV dUTPase,以為將 來分析其分子特性之用。 |
英文摘要 | Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dump and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. Previous work in our laboratory has cloned and sequenced the gene encoding the dUTPase of infectious bovine rhinotracheitis virus (IBRV) strain YL isolated in Taiwan. In this study, the whole coding region of the IBRV dUTPase gene was positioned downstream of the phage T7 promoter in the expression vector pET-28b(+). The resulting recombinant plasmid, pET/HJ4, was then transformed into Escherichia coli BL21(DE3)[pLysS] cells, conferring production of IBRV dUTPase as 1-2% of the soluble protein inducible by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluble extracts were passed over a nickel-chelating affinity chromatographic column followed by dialysis and ultrafiltration to obtain an enriched protein fraction. The purified product gave a single 40-kDa band on sodium dedecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and displayed a specific activity of converting dUTP to dUMP (41 μmol per minute per milligram of protein). The expression and purification scheme presented here provides and easily accessible source of IBRV dUTPase for protein engineering studies on its structure and function relationship. |
本系統中英文摘要資訊取自各篇刊載內容。