microRNA-140真核表达载体的构建及鉴定

摘要

Objective To construct a eukaryotic expression vector of microRNA-140(miR-140)and detect its expression in rabbit articular chondrocytes in vitro.Methods MiR-140 amplified from human genomic DNA isolated from whole blood was digested and inserted into pBudCE4.1 vector to generate pBudCE4.1-miR-140 eukaryotic expression vector.The nucleus localization signal linked nucleic kinase substrate short peptide (NNS) was conjugated to chitosan to form NNSCS,then NNSCS was respectively combined with pBudCE4.1-miR-140 and pBudCE4.1 vector to form NNSCS/pDNA complexes.NNSCS/pDNA complexes were transfected into primary rabbit articular chondrocytes in vitro.The expression of mature miR-140 was detected by using quantitative real-time PCR (qRT-PCR).Results MiR-140 was successfully cloned into pBudCE4.1 vector confirmed by restriction enzyme digestion and plasmid se-quencing.By qRT-PCR,the expression of miR-140 was significantly increased by about 14.5 times in NNSCS/pBudCE4.1-miR-140 transfection group when compared with NNSCS/pBudCE4.1 transfection control group (P ＜ 0.05).Conclusion The recombinant eukary-otic expression vector pBudCE4.1-miR-140 is successfully constructed.qRT-PCR results showed that the mature miR-140 was ef-fectively expressed in transient transfection of articular chondrocytes.The study lays a foundation for exploring the role of miR-140 in the repair of cartilage injury in the future.%目的 构建miR-140真核表达载体并检测其在兔软骨细胞中的表达情况.方法 以人全血标本基因组为模板,PCR扩增出带有酶切位点的miR-140序列,将该序列定向克隆入pBudCE4.1载体中,构建pBudCE4.1-miR-140真核表达载体.将核定位信号肽偶联核激酶底物短肽(nucleus localization signal linked nucleic kinase substrate short peptide,NNS)修饰的壳聚糖(NNSCS)分别与pBudCE4.1-miR-140重组质粒及pBudCE4.1空质粒形成纳米复合物,分别瞬时转染体外分离培养的正常兔原代关节软骨细胞,实时荧光定量PCR方法检测miR-140的表达情况.结果 构建的pBudCE4.1-miR-140真核表达质粒酶切鉴定和测序结果均正确,表明miR-140成功克隆入pBudCE4.1载体中.实时荧光定量PCR结果表明,与NNSCS/pBudCE4.1对照组相比,NNSCS/pBudCE4.1-miR-140转染组软骨细胞中miR-140表达升高约14.5倍(P＜0.05).结论 成功构建了pBudCE4.1-miR-140真核表达载体,瞬时转染后软骨细胞可以有效地高表达miR-140,为将来探究miR-140在软骨损伤修复中的作用机制奠定了基础.