摘要
[Objective] The study aimed at cloning PKR gene from Ctenopharyngodon idellus induced by PolyI：C in vitro,so as to provide foundation for study on the anti-virus genes of C.idellus.[Method] By referring to the PKR gene sequences of zebra fish（AJ852023.1） and Carassius auratus（AY293929.1） in Genbank,three pairs of degenerate primers were designed with 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 not from the cells treated for 12 h;in addition,the expression level increased with the processing time.Part of the amplified sequence of C.idellus shared the homology of 100% and 81.48% with the sequences of carp and zebra fish separately.[Conclusion] Part of the PKR gene sequence was cloned successfully from C.idellus.Moreover,we have proved that PolyI：C induction is effective for PKR protein expression,which will provide reference for treating viral diseases of C.idellus.
[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。
基金
Supported by National Natural Science Foundation of Zhejiang Province (Y3110432 )
Huzhou Teachers College Science ResearchFoundation (2010YZ48)~~
作者简介
李景芬（1977-），女，浙江湖州人，讲师，博士，从事动物遗传育种的研究，E—mail：ljingfen@hutc.zj.cn。通讯作者。
