Cloning, Squencing, and Expression of the Infectious Bovine Rhinotracheitis Virus Uracil-DNA Glycosylase(UL2)Gene

• Main Authors: Hsu, Wei-Li, 徐維莉
• Other Authors: Chung, Yang-Tsung
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/49706303223612543443

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 85 === DNA尿嘧啶醣甘酵素(uracil-DNA glycosylase)簡稱UDGase，是細胞 執行DNA修補作用的重要酵素之一。此酵素廣泛存在於自然界各種生物體 ，且包括皰疹病毒。牛傳染性鼻氣管炎病毒(infecious bovine rhinotracheitis virus; IBRV)隸屬於Alphaherpesvirinae病毒亞科，其 基因體之單長區(unique long region)核甘酸序列分析顯示此區段包含有 一長615個核甘酸的開讀框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，我自本實驗室先前已製備得之IBRV(台灣YL株) 病毒基因體DNA中選殖出含有UL2開讀框的DNA片段，並將之轉接於原核表 現載體pET-28b(+)或pET-32b(+)中，然後送入大腸桿菌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。我接著針對具有較佳活性的21-kDa融合型IBRV UDGase蛋白進行系統性生化特性分析，結果顯示UDGase酵素屬於熱不安定 型蛋白質(thermolabile protein)，當10分鐘前處理的溫度高於37oC時， 其活性會隨著溫度上昇而下降。在所測試的酸鹼值範圍內(pH 6.0 ~ 10)，此酵素在pH 8.0時有最佳的反應活性。單價離子如鈉、鉀離子，分 別於濃度高於60 mM以及30 mM時，對UDGase酵素具有明顯抑制作用，而雙 價離子鎂、鈣、錳、鋅離子只要濃度達10 mM即可使其活性喪失殆盡。此 外，當反應緩衝液中所含螯合物質(chelating agent)EDTA的濃度高於10 mM時，或添加源自肉毒桿菌(Bacillus subtilis)噬菌體PBS 1之UDGase抑 制蛋白時均會嚴重影響及酵素活性。 Uracil-DNA glycosylase (UDGase) is an essential component of the cellular DNA repair machinery. It is present in a wide range of organisms, including herpesvirus. 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 IBRV UL2 protein for biochemical characterization, the DNA segment containing the UL2 sequence was cloned and inserted into the prokaryotic expression vector pET28b(+) or pET32(+) followed by transformation into Escherichia coli (BL21)(DE3)[pLysS] cells. Upon induction with isopropyl-β-D- thiogalactopyranoside(IPTG) these cells produced a 21-kDa and 40-kDa fusion protein, respectively. A column chromatographic procedure using metal affinity resins resulted in homogenous preparations of those proteins. Both of the purified fusion proteins exhibited specific UDGase activities in an in vitro enzyme assay,but the detectable activity of the protein derived from pET28b(+) was tenfoldhigher than that of another. Subsequent biochemical studies were then carriedout by using the 21- kDa recombinant UDGase. As the results show, this enzymeis a thermolabile protein. When the temperature of the 10-minute preincubation time was higher than 37oC, the activity of the enzyme was dramatically decreased. The enzyme was optimally active at pH8.0 and exhibited UDGase activity in the absence of any added metal ion. Addition of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, manganese chloride, or zinc sulfate to the assay mixture caused inhibition. Moreover, the chelating agent ethylenediamine tetraacetic acid (EDTA), when its concentration was higher than 10 mM, and the uracil glycosylase inhibitor (UGI) of Bacillus subtilis bacteriophage PBS 1 both inhibited the activity of the IBRV UDGase.