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題名 | 牛傳染性鼻氣管炎病毒去氧尿核甘三磷酸鹽水解酵素之缺損變異分析=Deletion Mutagenesis of the Infectious Bovine Rhinotracheitis Virus dUTPase: Effects on Its Enzyme Activity |
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作者 | 鍾楊聰; 王斐; 徐維莉; | 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115-124 |
分類號 | 437.22 |
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; 重組蛋白質; 缺損變異分析; Infectious bovine rhinotracheitis virus; dUTPase; Recombinant protein; Deletion mutagenesis; |
語文 | 中文(Chinese) |
中文摘要 | 去氧尿核廿三磷酸鹽水解酵素(dUTPase)普遍存在於自然界,在細胞之核廿酸 代謝與DNA複製過程中扮演著重要的角色。為探討此酵素結構與功能之關係,我們先前選殖 了台灣分離YL株牛傳染性鼻氣管炎病毒(IBRV) dUTPase基因,並將該基因的完整轉譯序列 (coding sequence;轉譯產物長325胺基酸)嵌入表現載體pET-28b(+),在大腸桿菌BL2l (DE3)[pLysS]細胞中做表現。此大量表現的重組蛋白質,在其N-端帶有來自表現載體的六個 組胺酸標記(histidine tag),可以鎳離子親和性層析法(nickel-chelating affinity chromatography)快速純化為均質。以此方法純化得的重組dUTPase經功能測試,顯示其具 有酵素活性。已發表之其他物種(包括大腸桿菌、酵母菌、果蠅細胞、人類細胞、反轉錄病 毒、痘病毒、皰疹病毒等)的dUTPase在其胺基酸序列中皆具有五個保留性區段 (conserved motifs);IBRV dUTPase分子的五個保留性區段分別位於胺基酸103-111、 172-182、217-220、256-262、315-323。在本報告中,我們針對保留性區段所在之位置 選用了適當的限制酵素進行dUTPase缺損基因(truncated gene)的構築。經蛋白質表現與純 化之後,由酵素活性測試結果得知,N-端21個胺基酸缺損者仍具有絕大部分活性,但另外三 種變體蛋白(胺基酸105-149、192-234、271-325之缺損)的活性則完全喪失。本實驗之 結果對未來關於lBRVdUTPase分子特性深一層的研究,提供了一個值得參考的指標。 |
英文摘要 | Deoxyuridine 51-triphosphate nucleotidhydrolase (dUTPase) is widespread in nature and plays important roles in nucleotide metabolism and DNA replication. TO study the structure and function relationship of this enzyme, we have previously cloned the gene encoding the dUTPase of infectious bovine rhinotracheitis virus (IBRV), inserted its entire coding sequence into the expression vector pET-28b(+), and expressed the protein in Escherichia coli BL21(DE3)[pLysS] cells. The overproduced recombinant protein, carrying a six-histidine tag at its N-terminal end, could be purified to apparent homogeneity by using one-step nickel-chelating affinity chromatography. The purified recombinant dUTPase exhibited enzymatic activity in an in vitro functional assay. It has been found that five highly conserved motifs are present in the amino acid sequences of dUTPase from various sources, including bacteria, yeast, Drosophila, human, retroviruses, poxviruses, and herpesviruses. In the IBRV dUTPase molecule, the motifs are located in amino acid residues 103-111, 172-182, 217-220, 256-262, and 315-323. In this report, based on the positions of these motifs we chose appropriate restriction enzymes to construct the in-frame deletions of the IBRV dUTPase gene, and then carried out gene transfer experiments to obtain sufficient amounts of pure mutant proteins. The subsequent dUTPase enzymatic assay showed that the protein with the amino acid deletion 1 to 21 retained most of the wide-type dUTPase gene, while the other three proteins, with the amino acid deletions 105 to 149, 192 to 234, and 271 to 325, completely lost their catalytic abilities. These results present the important relationship of conserved motifs to dUTPase activities and build up the foundation for detailed studies on molecular properties of the IBRV dUTPase. |
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