大鼠microRNA-327重组腺病毒载体的构建及其在心肌细胞的转染效率

目的:构建含微小RNA-327(miRNA-327)基因的腺病毒载体,并观察其在H9C2心肌细胞中的转染效率。方法:利用聚合酶链反应(PCR)法钓取目的基因miRNA-327,并将目的基因与穿梭载体GV202连接形成miRNA-327腺病毒表达载体。经酶切及测序鉴定后,将构建的miRNA-327腺病毒表达载体与包装质粒共转染到人胚肾293细胞,再通过Cre/loxP重组酶系统以获得重组腺病毒,并对其进行扩增、纯化及滴度测定。最后将构建成功的重组腺病毒载体转染H9C2心肌细胞48h,并通过荧光显微镜以及流式细胞仪测定其转染效率。结果:双酶切与测序结果证明了大鼠miRNA-327腺病毒载体构建成功,且最终获得滴度为2×10^10PFU/ml的病毒液。转染心肌细胞48h后倒置荧光显微镜观察结果显示,腺病毒转染效率为90.15%±5.15%,流式细胞仪检测结果显示其转染效率为85.46%±3.08%。结论:成功构建了含miRNA-327基因的重组腺病毒载体,其在H9C2心肌细胞中具有较高的转染效率。

Construction of rat recombinant microRNA-327 adenovirus vector and its transfection efficiency in rat myocardium

Objective:To construct the rat miRNA-327 adenovirus vector and observe its transfection efficiency in H9C2 cells.Methods:The target miRNA-327 was fished by polymerase chain reaction(PCR)and linked to the digested GV202 to form the adenovirus expression vector of miRNA-327.After enzymatic digestion and sequencing,the recombinant shuttle vector miRNA-327 and auxiliary packaging plasmids were transfected into the human embryonic kidney cells 293(HEK293),and then the recombinant adenovirus vector was obtained by using the Cre/loxP recombinant enzyme system.The obtained recombinant adenovirus vector was ultimately transfected into H9C2 myocardial cells,observed under the fluorescence microscope and its transfection efficiency was evaluated by flow cytometry.Results:The rat recombinant adenovirus vector miRNA-327 was successfully constructed with a titer of 2×10^10 PFU/ml by enzymatic digestion and DNA sequencing.After 48 h transfection,the cells emitted green fluorescence at a rate of 90.15%±5.15%and 85.46%±3.08%according to the fluorescence microscopy and flow cytometry respectively.Conclusion:This study successfully constructs miRNA-327 recombinant adenovirus vector,which has high transfection efficiency in H9C2 cells.