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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。
OBJECTIVE: To construct the specific miRNA and its expression vector pcDNATM6.2-GW / EmGFPmiR against rat Efnb2 gene (target protein EphrinB2), and to detect the interference of Efnb2 gene. Methods: According to the sequence of NM001107328, four pairs of miRNA oligo were designed and synthesized. Four pairs of oligo were annealed to double strand. Then four pairs of double-stranded miRNA oligo were inserted into the miRNA expression vector pcDNATM6.2-GW / EmGFPmiR respectively using the vector construction kit for cloning. Four miRNA expression plasmids were constructed, and the constructed pcDNATM6 The 2-GW / EmGFPmiR interference plasmid was transfected into rat pheochromocytoma PC12 cells, and the changes of Efnb2 gene expression were detected by qPCR. Results: DNA sequencing analysis proved that miRNA vector of Efnb2 gene was constructed correctly, and RT-PCR results showed that the expression of Efnb2 gene was significantly reduced. Conclusion: The miRNA vector targeting Efnb2 gene was successfully constructed and the Efnb2 gene was silenced after transfection.