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[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、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,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。
[Objective] The research aimed to clone the PKR gene of grass carp and lay the foundation for the research on the anti-viral gene of grass carp. [Method] According to the PKR gene sequences of zebrafish (AJ852023.1) and crucian carp (AY293929.1) on GenBank, three pairs of degenerate primers were designed by using Primer Premier 5.0 software. Grass carp and kidney were treated with 100μg / ml PolyI: C in vitro (Ctenopharyngodon indellus kidney cells, CIK) for 12,36 and 48 hours, and the total RNA of the treated cells was extracted. After reverse transcription, the PKR gene in the three treatment time cells was amplified by the falling PCR method. [Result] The PKR gene was not amplified after 12 h of treatment, and PKR gene was amplified at 36 and 48 h after treatment, and the expression of PKR gene was increased with the increase of treatment time. The amplified partial sequences were similar to those of crucian carp and zebrafish The sequence homology was 100.00% and 81.48% respectively. [Conclusion] The partial sequence of PKR gene in grass carp was successfully obtained. Poly I: C efficiently induced the expression of PKR protein in grass carp, which will help to create a new idea for the treatment of grass carp virus disease.