【摘要】 目的:构建靶向NBS1基因的microRNA真核表达载体,鉴定其转染宫颈癌细胞株Hela后的生物活性。方法:根据NBS1mRNA序列设计合成4对pre-microRNA片段,定向克隆到pcDNA6.2-GW/EmGFP-miR真核表达载体上,并将其转染至Hela细胞株中。采用菌落PCR和测序分析鉴定插入序列的完整性;采用实时荧光定量PCR和Westernblot检测鉴定重组体对NBS1基因表达的干扰效果以确定其生物活性。结果:构建的4组重组体插入片段的碱基序列完全正确。重组体能干扰Hela细胞NBS1基因的表达,4组重组体NBS1基因mRNA的表达水平和蛋白表达水平均降低,其中NBS1mi-2组的表达水平最低。结论:构建的4组NBS1microRNA重组体在Hela细胞株中都具有生物活性,且NBS1mi-2组的干扰作用最强。载体构建成功,为应用microRNA靶向NBS1的肿瘤基因治疗的研究奠定了基础。更多还原

【Abstract】 Objective:To construct NBS1 microRNA expressing eukaryotic recombinants,and identify biological activity of recombinants in Hela cell after transfection.Methods:According to sequence of NBS1mRNA,the NBS1 pre-microRNA was designed and synthesized,then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cell line.To detect integrity of inset fragment through colony PCR and sequencing analysis.The biological activity of recombinants through identify interference efficiency of NBS1 microRNA recombinants by way of Real-Time PCR and Western blot were determined.Results:Sequences of inset fragment in four microRNA expressing recombinants were correct.NBS1 mRNA and protein expression of four microRNA recombinants were decrease,which is the lowest in the NBS1mi-2 group.Conclusion:Four NBS1-targeting microRNA expressing recombinants all have biological activity in Hela cell line,and NBS1mi-2 recombinant has the most interference efficiency.The microRNA expressing plasmid which were successfully constructed and lay foundation for the studies on the tumor gene therapy of microrna targeting NBS1.更多还原