【摘要】 目的:针对大鼠的Efnb2基因序列(目的蛋白EphrinB2),构建特异性干扰微小RNA(miRNA)及其表达载体pcDNATM6.2-GW/EmGFPmiR,并检测其对Efnb2基因的干扰作用。方法:根据NM001107328基因序列设计并合成4对miRNA oligo;将4对oligo退火成双链。然后用载体构建试剂盒进行重组克隆,将4对双链miRNA oligo分别插入到miRNA表达载体pcDNATM6.2-GW/EmGFPmiR中,构建4个miRNA表达质粒,通过脂质体2000将已经构建好的pcDNATM6.2-GW/EmGFPmiR干扰质粒转染至大鼠嗜铬细胞瘤PC12细胞中,通过qPCR检测Efnb2基因表达水平的变化。结果:DNA测序分析证明Efnb2基因的miRNA载体构建正确,RT-PCR结果表明Efnb2基因的表达水平明显降低。结论:成功地构建了针对Efnb2基因的miRNA载体,转染细胞后对Efnb2基因沉默。更多还原

【Abstract】 Objective: To construct the specific and highly efficient interfering microRNA(miRNA) expression vector that can silence Efnb2 gene.Methods: Four pairs of miRNA oligo were designed and synthesized according to NM 001107328 gene sequences,annealed into a double strand.Four pairs of double-stranded miRNA oligo were inserted into the vector pcDNATM 6.2-GW/EmGFPmiR to construct four miRNA expression vectors.The pcDNATM 6.2-GW/EmGFPmiR vectors were used to transfect PC12 cells by lipofectmine 2000 reagent.The expression levels of mRNA were analyzed by qPCR.Results: The miRNA oligonucleotides of Efnb2 were correctly cloned into the pcDNATM6.2-GW/EmGFPmiR plasmids and confirmed by DNA sequencing.RT-PCR analysis revealed that the expression of Efnb2 mRNA in PC12 cells transfected with the pcDNATM6.2-GW/EmGFPmiR constructs of miRNA was significantly decreased as compared with that in the negative control PC12 cells.Conclusion: miRNA expression plasmids for silencing EphrinB2 protein gene are successfully constructed,and they effectively inhibit the Efnb2 gene expression.更多还原