摘要
根据鲫鱼类PKR蛋白激酶基因(CaPKR-like)的全长cDNA序列,克隆CaPKR-likeZα结构域cDNA(Zα1、Zα2和Zα1Zα2),原核表达成功获得3种融合蛋白PZα1、PZα2和PZα1Zα2。凝胶阻滞实验结果显示:PZα1、PZα2、PZα1和PZα2混合表达蛋白不能与聚肌胞苷酸(PolyI∶C)结合,而表达完整的Zα结构域的PZα1Zα2与PolyI∶C有明显的结合现象。另外,3种表达多肽PZα1、PZα2和PZα1Zα2在体外分别都能聚合形成二聚体。与PZα1相比,PZα2和PZα1Zα2二聚化现象显著。结果暗示病毒复制时产生的副产物dsRNA能与CaPKR-like蛋白的相关结构域结合从而调节其生理功能。
Three fusion peptides, P Zα1, P Zα2 and P Zα1Zα2 for Zα1 domain, Zα2 domain, and Zα1Zα2 domains of Carassius auratus PKR-like gene, respectively, were successfully expressed by a prokaryotic expression system and then purified by affinity chromatography. Gel mobility shift assay revealed that P Zα1Zα2 rather than P Zα1, P Zα2, and mixture of P Zα1 and P Zα2, was capable to bind to polyinosinic∶polycytidylic acid (Poly I∶C) in vitro. In addition, all of the three fusion peptides all could form dimer, with strong dimerization for P Zα2 and P Zα1Zα2 but a relative weak one for P Zα1. The results suggest that dsRNA, the by-product generated during virus replication in host cells, probably binds to the Zα domain of CaPKR-like and then regulates its physiological function.
基金
国家重点基础研究发展规划(973)项目(2004CB117403)
国家高技术研究发展计划(863)项目(2002AA626010)
国家自然科学基金资助项目(30200207
30471333)
江西省教育厅项目(0405)资助
