Chung Y T, Hsu W
Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan.
Microbiol Immunol. 1996;40(12):949-53. doi: 10.1111/j.1348-0421.1996.tb01164.x.
Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence down-stream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl beta-D-thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.
先前对牛疱疹病毒1型（BHV-1）基因组独特长片段的序列分析鉴定出一个开放阅读框（ORF），命名为UL2，其推导的204个氨基酸的多肽包含一个共有尿嘧啶-DNA糖基化酶（UDGase）特征序列。为了确定BHV-1 UL2 ORF产物是否具有UDGase活性，我们将UL2序列定位在载体pET-28b(+)上T7启动子的下游，并在大肠杆菌中进行表达。用异丙基-β-D-硫代半乳糖苷诱导后，这些细胞产生了一种23 kDa的蛋白质，其分子量与核苷酸序列预测的一致。使用镍螯合亲和色谱的一步纯化程序得到了该蛋白质的均一制剂，其在体外酶测定中显示出特异性UDGase活性。这些结果提供了证据，证明BHV-1 UL2基因确实编码一种UDGase。
