miRNA-140过表达腺病毒载体的构建及鉴定

The construction and identification of an adenoviral vector expressing mature miRNA-140

目的:构建稳定表达成熟miRNA-140腺病毒表达载体。方法:从人类基因组中扩增出带有酶切位点的miRNA-140目的基因,将目的基因连接到穿梭质粒p DC316-m CMV-EGFP,进一步将带有目的基因的穿梭质粒重组到骨架质粒Ad Easy,并将重组质粒转染到AAV-293细胞中包装成腺病毒载体,经过二次扩增之后获得高滴度的腺病毒,并且检测其包装效率及感染滴度。最后,用目的腺病毒感染骨肉瘤细胞,通过荧光定量PCR方法检测miRNA-140表达情况。结果:酶切鉴定和测序结果均表明,miRNA-140成功克隆入p DC316-m CMV-EGFP载体中。与Ad Easy重组后,包装纯化具有感染性的腺病毒miRNA-140,通过荧光定量PCR检测,软骨肉瘤细胞中miRNA-140升高4.2±0.2倍。结论:成功构建了成熟miRNA-140的腺病毒表达载体。

Objective：To construct an adenoviral vector expressing mature miRNA-140. Methods：The target Hsa-miRNA-140 gene amplified from human genome was digested and linked to the shuttle plasmid pDC316-mCMV-EGFP. The recombinant plasmid was confirmed and transfected into AAV-293 cells for adenoviruses pAd-has-miRNA-140 packaging. The obtained adenoviruses were used to infect target cells and the cellular expressions of has-miRNA-140 were detected using fluorescence and quantitative PCR. Results：Has-miRNA-140 containing the restriction sites was amplified and linked to the shuttle plasmid pDC316-mCMV-EGFP,which was successfully recombined with AdEasy.After packaging in AAV-293 cells,the adenoviruses were obtained,which caused an increase of miRNA-140 expression about 4.2±0.2 folds in osteosarcoma cells. Conclusion：The adenoviral vector expressing the mature miRNA-140 is successfully constructed.