蛋白激酶PKR在HeLa细胞中的过表达及其siRNA干扰的鉴定  

Over expression of protein kinase PKR in HeLa cells and the identification of its siRNA interference

作　　者：吴娜[1] 赵志荀[1] 吴国华[1] 颜新敏[1] 叶奕优[2] 李应国[3] 朱海霞[1] 李健[1] 张志东[1] 张强[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,兰州730046 [2]深圳出入境检验检疫局动植物检验检疫技术中心,广东深圳518045 [3]重庆出入境检验检疫局,重庆400020

出　　处：《黑龙江畜牧兽医》2016年第5期43-47,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31201892);"863"国家高技术研究发展计划项目(2012AA101304);国家质检总局公益性行业科研专项(201310093);甘肃省自然科学基金项目(1208RJZA101)

摘　　要：为了研究双链RNA依赖的蛋白激酶(PKR)信号通路对羊痘病毒复制的影响,试验构建了PKR基因的真核表达载体,并在He La细胞中实现了PKR的过表达;从相关文献中筛选了靶向PKR的2对siRNA,检测其对PKR的干扰效果,以RT-PCR方法扩增PKR基因,克隆入表达载体pc DNA3.1(-)/Myc-His(B),对重组质粒进行双酶切及测序鉴定,将阳性重组质粒或siRNAs分别转染He La细胞,Western-blot检测PKR蛋白的表达水平。结果表明:试验成功构建了PKR基因的真核表达载体pc DNA3.1(-)-PKR,重组质粒在He La细胞中得到了表达;2对siRNA具有较好的干扰效果,其中siRNA1、siRNA2对PKR的干扰效率分别达到76%和52%。To study the double -stranded RNA dependent protein kinase （PKR） signal pathway on capripexvirus replication, a eukaryotic expression vector of PKR gene was constructed, and the PKR was over - expressed in HeLa cells. Two pairs of siRNAs of targeting PKR were screened and used for detecting the effect of PKR interference. The PKR gene was amplified by RT - PCR, and cloned into the expression vector pcDNA3.1 （ - ）/Myc - His （ B ）. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. Positive recombinant plasmids and siRNAs were transfected into HeLa ceils respectively, and the expression level of PKR protein was detected by Western - blot. The results showed that the eukaryotic expression vector pcDNA3.1 （ - ） - PKR of PKR gene was successfully constructed, and the recombinant plasmids were expressed in HeLa ells. The two pairs of siRNAs had better interference effect, thereinto, the interference efficiency of siRNA 1 and siRNA2 to PKR reached 76% and 52% , respectively.

关 键 词：羊痘病毒 PKR基因 真核表达 SIRNA HELA细胞 

分 类 号：S852.654[农业科学—基础兽医学] Q784[农业科学—兽医学]