【摘要】 目的构建促血管生成素2(Ang-2)及其受体Tie2基因的RNA干扰(RNAi)表达载体。方法以Ang-2、Tie2的mRNA已知序列为靶点,化学合成能编码针对Ang-2、Tie2基因的短发夹RNA(shRNA)序列的寡核苷酸各2对,退火处理后克隆到pSilencer1.0-U6-siRNA表达载体的U6RNA聚合酶Ⅲ启动子的下游,构建重组质粒pSliencer-1.0-U6-Ang-2/Tie2-siRNA。结果将与目的片段相符的双链shRNA和线性化的pSilencer1.0-U6-siRNA质粒连接后,经限制性内切酶HindⅢ酶切电泳及测序分析证实,2条重组质粒载体构建正确,无任何碱基突变。结论成功构建pSilencer1.0-U6-Ang-2/Tie2-siRNA重组质粒各2条,为进一步研究其对血管生成的作用打下基础。更多还原

【Abstract】 Objective To construct the RNAi expression vector for angiogenin-2(Ang-2)and Tie2 genes.Methods By using the mRNAs of Ang-2 and Tie2 as the target sequences,two pairs of oligonucleotides which encoded short hairpin RNA(shRNA)of Ang-2and Tie2 genes respectively were synthesized with chemical way.After annealing,they were cloned into the downstream of U6RNA polymerase Ⅲ(pol Ⅲ)promoter of psilencer1.0-U6-siRNA expression vector,and then the RNAi recombinant plasmids-psliencer-1.0-U6-Ang-2/Tie2-siRNA were constructed.Results By using enzyme digestion electrophoresis and sequencing analysis of the restriction endonuclease Hind Ⅲ,it was confirmed that the purpose sequence was successfully inserted into the expected site.Conclusion The recombinant plasmids psliencer-1.0-U6-Ang-2-siRNA-1,2 and psliencer-1.0-U6-Tie2-siRNA-1,2 of each gene were constructed successfully,and would be useful for the further research of angiogenesis.更多还原