新靶点CyclinE干扰RNA真核表达载体的构建

Construction of new target eukaryotic expression vectors of CyclinE siRNA

目的构建4个新靶点的人CyclinE干扰RNA真核表达载体,转染人脑胶质细胞瘤U251细胞,经反转录-聚合酶链反应(RT-PCR)检测mRNA表达,获得干扰效果最好的真核表达载体,为CyclinE成为人脑肿瘤标志物提供有价值的资料。方法 (1)构建4个新靶点CyclinE干扰RNA的真核表达载体后,用双酶切和碱基序列测定的方法鉴定载体构建成功与否;(2)用Lipofectamine 2000转染4个成功构建的载体到胶质瘤U251细胞株;(3)通过RT-PCR的结果检测转染后CyclinEmRNA表达量,选出干扰效果最好的1个。结果 (1)成功构建了4个新靶点的CyclinE干扰RNA真核表达载体即PCyclinE-1、PCyclinE-2、PCyclinE-3、PCyclinE-4。(2)CyclinEmRNA表达明显受到抑制,并获得效果最好的CyclinE干扰RNA真核表达载体。结论成功构建并筛选出效果最好的新靶点CyclinE干扰RNA真核表达载体,使U251细胞的CyclinE mRNA表达明显降低。

Objective To construct the 4new targets eukaryotic expression vectors of human CyclinE interference specific RNA to transfect into glioma U251 cells,the mRNA expression was detected by RT-PCR,for obtaining the ukaryotic expression vectors with the best interference effect to provide the valuable data for Cyclin E becoming the human tumor marker.Methods（1）Four new targets eukaryotic expression vectors of RNA interference specific CyclinE were constructed,named as PCyclinE-1,PCyclinE-2,PCyclinE-3 and PCyclinE-4.The double digestion method and the base sequence measuring were adopted to detect whether the vectors were successfully constructed.（2）The four newly constructed vectors were transfected into U251 cell line by Lipofectamine 2000.（3）The RT-PCR results was adopted to detect the expression quantity of CyclinE mRNA for selecting a vector with best interference.Results（1）4new targets eukaryotic expression vectors of RNA interference specific for CyclinE were constructed.（2）CyclinEmRNA was obviously suppressed,the eukaryotic expression vector with best interference effect for human CyclinE interference specific RNA was obtained.Conclusion The new eukaryotic expression vector of CyclinE siRNA with best effect is successfully constructed,which significantly suppress the CyclinE mRNA expression of U251 cells.