蓝舌病病毒非结构蛋白NS2单链RNA结合结构域的定位
Localization of the single-stranded RNA-binding domains of bluetongue virus nonstructural protein NS2.
作者信息
Fillmore G C, Lin H, Li J K K
机构信息
Department of Biology, Utah State University, Logan, Utah 84322-5305, USA.
出版信息
J Virol. 2002 Jan;76(2):499-506. doi: 10.1128/jvi.76.2.499-506.2002.
Abstract
The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed. Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein. A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations. These truncated proteins were expressed on a large scale and purified to near homogeneity. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays. As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.
摘要
蓝舌病病毒17型的S2基因已被克隆，非结构蛋白NS2已得到表达。利用与NS2氨基酸序列内区域匹配的合成肽来定位该蛋白内的三个单链RNA（ssRNA）结合区域。然后使用原核表达系统生成一系列缺失突变体，其中NS2的ssRNA结合结构域被单独或不同组合地去除。这些截短的蛋白被大规模表达并纯化至接近均一性。然后通过电泳迁移率变动分析测定每个截短蛋白对ssRNA的亲和力。结果，蓝舌病病毒非结构蛋白NS2的三个ssRNA结合结构域已被最终定位，去除这三个结构域完全消除了NS2与ssRNA结合的能力。
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