E2F-1基因RNAi慢病毒载体的构建与鉴定

目的：构建和鉴定靶向E2F-1基因的RNA干扰慢病毒载体。方法：体外合成两对互补并编码靶向E2F-1基因的特异性短发夹RNA（short hairpin RNA,shRNA）序列和一个阴性对照组的寡核苷酸,克隆到pLKO.1-PURO-U6（Age I/EcoR I）质粒载体中,经酶切和测序鉴定所构建的重组载体是否正确,将以上质粒分别和包装质粒混合物共转染293T细胞,包装产生病毒颗粒。各组病毒载体转染Tca8113细胞后,运用Real-Time PCR和Western检测三组E2F-1 mRNA和蛋白的表达水平。结果：经酶切和测序证明,pLKO.1-PURO-U6-E2F-1shRNA序列正确;转染后,各组慢病毒载体中,第一组质粒感染后的Tca8113细胞中E2F-1基因的核酸电泳条带明显减弱,Western检测出E2F-1蛋白的表达降低。结论：靶向E2F-1基因的shRNA慢病毒载体构建成功,并可特异性沉默E2F-1基因的表达,为研究E2F-1在口腔鳞癌的发生发展中的作用提供了初步的实验基础。

Construction and identification of lentiviral vector of RNA interference of E2F-1 gene

Objective： To construct a lentiviral vector-mediated RNA interference（RNAi） of targeting gene E2F-1.Methods： Two short hairpin RNA oligonucleotides targeting gene E2F-1 on appropriate site and one pair of negative control oligonucleotide sequence were synthesized and inserted into pLKO.1-PURO-U6（Age I/EcoR I） vector.The sequences of three plasmids were identified by DNA sequencer and restriction endonuclease digestion.These recombinant plasmids were transfected into the 293T cells along with lentiviral packing mix by lipofectamine 2000 for the package of lentiviral particles.Then the lentiviral vector particles were transfected into Tca8113 cells and E2F-1 expression in the transfected cells was assayed by Real-Time PCR and Western.Results： It was verified that the specific DNA oligonucleotide was cloned into the vector successfully.In the first group,the expression of E2F-1 mRNA and the expression of E2F-1 protein in Tca8113 cells was significantly reduced after transfecting the recombinant plasmids,compared to the control.Conclusions： It indicates the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of E2F-1 mRNA,and those provides the precondition for the further study of E2F-1 in OSCC（oral squamous cell carcinoma） pathogenesis by using lentivirus system to construct stably silencing E2F-1 in OSCC cell lines.