pcDU6质粒载体介导的表达TGF-β_1 sh RNA的质粒构建

Construction of Plasmid Expressing TGF-β_1 Sh RNA Mediated by pcDU6 Plasmid Vector

【目的】用 pcDU6质粒载体构建介导转化生长因子 β1(TGF β1)短发夹RNA(shorthairpinRNA ,shRNA)表达的质粒。【方法】针对TGF β1的以GGCC开始的 2 1碱基大小的片段 ,分别设计 2对寡核苷酸 ,形成双链后将其依次连入带有U6启动子的pcDNA3.1(- )载体 (命名为 pcDU6 ) ,两对DNA双链通过BamHⅠ酶切位点连接后形成中间由 6个碱基序列间隔的反向互补序列 ,构建成TGF β1短发夹RNA的产生质粒。【结果】经酶切、连接后构建成的质粒称之为 pcDU6 A1 A2和 pcDU6 B1 B2 ,经酶切与测序证实构建成功 ,无任何碱基突变。【结论】成功构建了能表达TGF β1shRNA的质粒载体 pcDU6 A1 A2和pcDU6 B1 B2 ,可能为临床防治长期腹膜透析病人的腹膜纤维化提供高效的方法。

To construct a plasmid expressing transforming growth factor β -short hairpin RNA (TGF-β Sh RNA) mediated by pcDU6 plasmid vector.Two selected fragments of coding sequence from TGF-β contained 21 nucleotides started with GGCC. Two pairs of oligonucleotides were designed for these two fragments. After annealing double stranded DNAs formed were ligated to plasmid pcDU6 [pcDNA3.1(-) vector with U6 promoter] separately. The plasmids producing TGF-β Sh RNA were constructed from the inverted motif contained 6 spacer and four Ts.The constructed plasmids were called pcDU6-A1-A2 and pcDU6-B1-B2, their successful construction was identified by endonuclease digestion and sequence analysis, without any base pair mutation. [Conclusion] Plasmid vectors pcDU6-A1-A2 and pcDU6-B1-B2 expressing TGF-β Sh RNA were successfully constructed, providing a highly effective method for prevention and treatment of peritoneal fibrosis in patients subjected to long-term peritoneal dialysis.