microRNA-140真核表达载体的构建及鉴定

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。

Construction and Identification of MicroRNA-140 Eukaryotic Expression Vector

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 sequencing. 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 eukaryotic expression vector pBudCE4.1-miR-140 is successfully constructed. qRT-PCR results showed that the mature miR-140 was effectively 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.