携带DREAM基因小分子干扰RNA重组质粒的构建

背景：DREAM是一种多功能蛋白，在细胞中不同位置与不同靶蛋白结合，体外细胞培养和动物实验均证明DREAM参与了许多疾病的发病机制。目的：构建携带DREAM基因的小分子干扰RNA重组质粒。方法：设计并合成shRNA对应的两条互补的寡核苷酸链，pDC316-EGFP—U6质粒经BamH I 和 HindIII双酶切与退火后的寡核苷酸连接，转化感受态coliDH5a，获得阳性克隆进行PCR和测序鉴定。结果与结论：经PCR、酶切及测序证实，重组质粒pDC316-EGFP．DREAM-shRNA-U6片段大小为473bp，其中插入的片断序列和位点与预期完全一致，说明pDC316．EGFPDREAM．shRNA-U6重组质粒构建成功。

Construction of recombinant DREAM-targeting small interfering RNA expressing plasmids

3ACKGROUND： DREAM is a multi-functional protein, which combine with different target proteins at different sites in cells. In vitro ：ultivate tests and animal experiments confirmed that DREAM is involving in onset mechanism of many different diseases. OBJECTIVE： To construct recombinant DREAM-targeting small interfering RNA （siRNA） expressing plasmids. METHODS： Oligonucleotide containing the small hairpin of DREAM was designed and synthesized, which was inserted into the ;DC316-EGFP-U6 plasmid double digested by BamH I and Hind III. The liation product was transformed competence E.coli DH5a. Positive clones were identified by PCR and sequencing. rESULTS AND CONCLUSION： The result of PCR and gene sequencing confirmed that the pDC316-EGFP-DREAMshRNA-U6 ecombinant plasmid with 473 bp had been constructed successfully.