Zhao S L, Liang C Y, Zhang W J, Tang X C, Peng H Y
Laboratory for Biological Control, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
Virus Res. 2005 Dec;114(1-2):80-8. doi: 10.1016/j.virusres.2005.06.002. Epub 2005 Jul 14.
Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV-1) belongs to the Cypovirus genus in the Reoviridae family. The ORF of genome segment 8 (S8) of DpCPV-1 was cloned into vector pMAL-c2X and used to express a 44kDa protein (p44) in E. coli, which was detected by Western blotting. The gel mobility shift assays showed that p44 had ssRNA-binding activity. Competitive assay indicated that this protein only bind to ssRNA and could not interact with DNA and dsRNA. The binding of p44 to ssRNA is sequence non-specific. To identify the domain(s) important for RNA binding of the protein, a number of deletions were made. These truncated proteins were expressed in E. coli and purified. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays and northwestern blot. The results indicated that glutamic acid-rich domain in the central region of p44 from residues 104 to 201 was the ssRNA-binding domain.
马尾松毛虫细胞质型多角体病毒（DpCPV-1）属于呼肠孤病毒科质型多角体病毒属。将DpCPV-1基因组片段8（S8）的开放阅读框克隆到载体pMAL-c2X中，并用于在大肠杆菌中表达一种44kDa的蛋白质（p44），通过蛋白质免疫印迹法检测到该蛋白。凝胶迁移率变动分析表明p44具有单链RNA结合活性。竞争分析表明该蛋白仅与单链RNA结合，不能与DNA和双链RNA相互作用。p44与单链RNA的结合不具有序列特异性。为了鉴定该蛋白RNA结合的重要结构域，进行了一系列缺失。这些截短的蛋白在大肠杆菌中表达并纯化。然后通过电泳迁移率变动分析和蛋白质印迹杂交法检测每种截短蛋白对单链RNA的亲和力。结果表明，p44中央区域104至201位残基富含谷氨酸的结构域是单链RNA结合结构域。
