靶向PTTG和survivin双干扰siRNA表达载体的构建及鉴定

Construction and identification of double interference vector targeting both PTTG and survivin gene

目的:构建同时干扰人垂体瘤转化基因(PTTG)和生存素(survivin)基因表达的RNA共干扰载体,并检测其对人脑胶质瘤U251细胞中PTTG基因和survivin基因的干扰作用。方法:根据GenBank数据库中PTTG和survivin的cDNA序列设计干扰序列,构建干扰PTTG和生存素基因的重组干扰质粒pGenesil-2.1-PTTG siRNA和pGenesil-2.1-survivin siRNA。酶切、测序鉴定正确后,将pGenesil-2.1-Survivin和pGenesil-2.1-PTTG质粒分别进行HindⅢ和BamHⅠ双酶切,1%琼脂糖凝胶电泳分别回收质粒survivin双酶切大片段和质粒PTTG双酶切小片段(约380bp),将质粒survivin回收大片段与质粒PTTG回收小片段进行连接,得到双干扰质粒pGenesil-2.1-PTTG-survivin siRNA,酶切鉴定,同时检测其对U251细胞PTTG和survivin基因mRNA表达的干扰作用。结果:测序和酶切鉴定结果显示,pGenesil1-PTTG、pGenesil1-survivin和双干扰质粒pGenesil2.1-PTTG-survivin siRNA构建正确;RT-PCR结果显示,将双干扰质粒转染U251细胞48h后,U251细胞中PTTG基因和survivin基因mRNA表达明显降低。结论:成功构建了能同时干扰PTTG和survivin基因的双干扰pGenesil1-PTTG-Survivin siRNA表达载体,并可有效抑制U251细胞PTTG和srvivin基因mRNA的表达。

Objective To construct the double interference vector targeting both PTTG and survivin gene and detect its silencing effects on PTTG and survivin gene in human glioma U251 cell line.Methods Two effective targeting sequences were chosen according to PTTG and survivin cDNA sequences in GenBank,then they were inserted into pGenesil-2.1 vector and pGenesil-2.1-PTTG and pGenesil-2.1-survivin plasmids were constructed.The recombinant plasmids were identified by restriction endonuclease and DNA sequencing.pGenesil-2.1-survivin and pGenesil-2.1-PTTG plasmids were digested by HindⅢ and BamHⅠ separately,long fragment of pGenesil-2.1-survivin plasmid and small fragment of pGenesil-2.1-PTTG(about 380 bp) were collected by 1% agarose gel electrophoresis.The double interference vector pGenesil-2.1-PTTG-survivin siRNA was constructed by uniting long fragment of pGenesil-2.1-srvivin plasmid and small fragment of pGenesil-2.1-PTTG.This plasmid was identified by restriction enzyme digestion and then its silencing effects on PTTG and surviving gene were detected.Results By restriction endonuclease and DNA sequencing,the eukyaryotic expression plasmid of pGenesil-2.1-PTTG,pGenesil-2.1-survivin and pGenesil-2.1-PTTG-survivin siRNA were constructed correctly.The result of RT-PCR showed that PTTG and survivin gene mRNA levels were decreased obviously after this plasmid was transfected into U251 cells for 48 h.Conclusion pGenesil-2.1-PTTG-survivin siRNA plasmid is constructed successfully.The PTTG and survivin gene mRNA levels in U251 cells are decreased effectively by transfecting this plasmid into U251 cells.