PolyI：C体外诱导草鱼PKR基因的克隆与表达

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100μg/ml PolyI：C体外分别处理草鱼肾细胞（Ctenopharyngodon indellus kidney cells,CIK）12、36、48 h,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12 h时未扩增出PKR基因,处理36和48 h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,PolyI：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。

Cloning and Expression of PKR Gene of Ctenopharyngodon idellus Induced by PolyI:C in Vitro

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 Genbank,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.