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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。
According to the CaPKR-like cDNA sequence of Carassius auratus, three CaPKR-likeZα domain cDNAs (Zα1, Zα2 and Zα1Zα2) were cloned, and three fusion proteins PZα1, PZα2 and PZα1Zα2 were successfully obtained. The result of gel retardation showed that the mixed expression of PZα1, PZα2, PZα1 and PZα2 could not bind to poly (Cy) I (PolyI: C), while PZα1Zα2, which expressed intact Zα domain, had obvious binding with PolyI: C . In addition, the three expressed polypeptides, PZα1, PZα2 and PZα1Zα2, were able to polymerize in vitro to form dimers, respectively. Compared with PZα1, PZα2 and PZα1Zα2 dimerization phenomenon is significant. The results suggest that dsRNA, a byproduct produced during virus replication, binds to the related domains of CaPKR-like proteins to regulate its physiological function.