靶向人 BRG1基因的慢病毒载体构建及效率验证

目的：构建靶向人 BRG1基因的短发夹 RNA（shRNA）慢病毒载体并验证其沉默效率。方法：设计3个针对人 BRG1基因的特异性 shRNA 序列（shBRG1），构建于 pLKO．1慢病毒载体中，并与 psPAX2和 pMD2．G 质粒共同转染293T 细胞以包装成慢病毒颗粒。将包装好的慢病毒感染人主动脉平滑肌细胞（HASMC），应用实时荧光定量 PCR 和 western blot 检测 BRG1 mRNA 和蛋白表达水平，判断其沉默效率。结果：3种 shBRG1干扰序列均成功插入慢病毒载体中且测序正确，3种慢病毒均可有效降低 BRG1 mRNA 表达水平（P 均＜0．01）。其中 shBRG1-3的沉默效率最高，其感染HASMC 后 BRG1蛋白表达水平较对照组显著下降（P ＜0．01）。结论：靶向人 BRG1基因的 shRNA 慢病毒表达载体构建成功，shBRG1-3慢病毒能够有效降低 BRG1 mRNA和蛋白表达水平。

Construction of lentiviral vector targeting human BRG1 gene and identification of its efficiency

Objective：To construct short hairpin RNA （shRNA）lentivirus targeting human BRG1 gene and detect its efficiency of gene silence in human aortic smooth muscle cells （HASMC）. Methods：Three specific shRNA sequences targeting human BRG1 gene （shBRG1 ）were cloned into pLKO.1 lentiviral vector respectively,and pLKO.1-shBRG1 was transfected into 293T cells with psPAX2 and pMD2.G.Lentiviral particles produced by 293T cells were then used to infect HASMC.Silence efficiency of BRG1 mRNA and protein was determined by qRT-PCR and western blot after infection. Results：It was confirmed by DNA sequencing that shBRG1 sequences were correctly inserted into the pLKO.1 lentiviral vector.All three shBRG1 lentivirus effectively downregulated the expression of BRG1 mRNA in HASMC,and shBRG1-3 had the highest silencing efficiency （P〈 0.01 ）.BRG1 protein expression was significantly downregulated after infection of shBRG1-3 in HASMC compared with negative control （P 〈;0.01 ）. Conclusion：Human BRG1 gene RNA interference lentivirus have been successfully constructed. The expression levels of BRG1 mRNA and protein can be significantly downregulated in HASMC when infected with shBRG1-3 lentivirus.