PolyⅠ:C体外诱导草鱼PKR基因的克隆与表达

摘要

[目的]克隆草鱼的PKR基因,为草鱼抗病毒基因研究奠定基础.[方法]依据GenBank上斑马鱼(AJ852023.1)和鲫鱼(AY293929.1)的PKR基因序列,利用Primer Premier 5.0软件设计了3对简并引物；采用100 μg/ml Polyl:C体外分别处理草鱼肾细胞(Ctenopharyngodon indellus kidney cells,CIK)12、36、48h,并提取处理后细胞的总RNA,逆转录后用降落PCR方法扩增这3个处理时间细胞中PKR基因.[结果]处理12h时未扩增出PKR基因,处理36和48h时都扩增到了PKR基因,并且表达量随处理时间的增加有所升高,扩增到的部分序列与鲫鱼和斑马鱼的该段序列同源性分别为100.00％和81.48％.[结论]试验成功获得了草鱼PKR基因的部分序列,Polyl:C高效诱导草鱼PKR蛋白表达将有助于开创治疗草鱼类病毒病的一种新思路.%[Objective] The study aimed at cloning PKR gene of Ctenopharyngodon idellus induced by PolyI: C in vitro, so as to provide foundation for study on the anti-virus genes of C. Idellus. [ Method] On the basis of the PKR gene sequences of zebra fish (AJ852023.1) and carp ( AY293929.1) in Cenbank, three pairs of degenerate primers were designed using Primer Premier 5.0 software; in vitro C. Idellus kidney cells (CIK) were treated with 100 μg/ml of Poly Ⅰ: C for 12, 36 and 48 h, and then total RNA of the cells treated was extracted for amplifying the PKR gene by RT-PCR. [Result] The PKR gene was amplified from the cells treated with Poly Ⅰ:C for 36 and 48 h. But the cells treated for 12 h; in addition, the expression level increased with the processing time. The amplified partial sequence of C. Idellus shared homology of 100.00% and 81.48% with carp and zebra fish sequence separately. [Conclusion] Part of the PKR gene sequence was cloned successfully from C. Idellus, PolyⅠ:C induction was effective to PKR protein expression.