PTTG基因siRNA载体的构建与鉴定

目的构建针对垂体腺瘤细胞系AtT20的高效率沉默PTTG基因的shRNA表达载体。方法合成特异性干扰PTTG基因的小发卡RNA(shRNA)片段,并与pGenesil2载体连接,构建PTTG干扰载体(pGenesil2-PTTG shRNA)。利用脂质体将其转染AtT20细胞,分为正常细胞对照组、阴性对照组和3个shRNA干扰组(pGenesil2-PTTG siRNA1、2及3),应用半定量逆转录聚合酶链式反应(RT-PCR)和Western blot法分析转染后AtT20细胞中PTTG的mRNA和蛋白表达水平。结果酶切证实PTTG-shRNA表达载体构建成功;转染效率可达75%左右。转染pGenesil2-PTTG shRNA后,3个干扰组的AtT20细胞中PTTG的mRNA和蛋白表达水平均较正常对照组显著降低(P<0.01)。结论成功构建了能高效沉默PTTG的RNAi表达载体;且pGenesil2-PTTG siRNA高效地抑制了垂体腺瘤细胞AtT20细胞中PTTG綦因的表达。

Construction and identification of siRNA expression vectors for silencing PTTG gene

Objective To construct the specific high efficiency small interfering RNA(siRNA)expression vector that can silence PTTG gene.Methods Using vector based RNA interference technique,vectors were constructed to transcribe the functional short hairpinRNA(shRNA) specially targeting PTTG.The vectors were used to transfect AtT20 cells by lipofectamine2000 reagent.And the cells were divided into five groups:normal control,negative group and three siRNA interfering groups(pGenesil2-PTTG siRNA1,pGenesil2-PTTG siRNA2 and pGenesil2-PTTG siRNA3).The expression levels of mRNA and protein of PTTG were analyzed by RT-PCR and Western blotting methods.Results By restriction endonuclease and DNA sequencing analyzing,eukaryotic expression plasmid of PTTG was successfully constructed.PTTG mRNA and its protein level in transfected AtT20 cells with PTTG RNAi plasmid decreased significantly compared with the normal control group(P<0.01),and the transfection rate reached approximately 75%.Conclusion pGenesil2-PTTG siRNA vectors were successfully constructed,and they can silence the PTTG gene in AtT20 cells with high efficiency.