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pDC316-mCMV-EGFP,进一步将带有目的基因的穿梭质粒重组到骨架质粒AdEasy,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pDC316-mCMV-EGFP载体中。与AdEasy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。 |
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