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

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.