miR-137过表达慢病毒的包装和鉴定

目的：构建miR-137过表达慢病毒载体并进一步包装慢病毒，探讨miR-137在HEK293T细胞中的感染效率和表达水平。方法：化学合成miR-137序列并克隆到慢病毒载体GV209，得到并鉴定含有目的片段的重组质粒，采用Lipofectamine 2000共转miR-137重组质粒与慢病毒辅助包装质粒到HEK293T细胞中生产慢病毒。以感染复数（MOI）值为40感染HEK293T细胞48h后，荧光显微镜下观察细胞中绿色荧光蛋白（GFP）的表达，采用荧光定量PCR法检测HEK293T细胞中miR-137的表达水平。结果：测序结果，目的基因序列与GenBank公布的miR-137基因序列完全一致。在感染的HEK293T细胞中观察到GFP的表达。过表达慢病毒感染细胞中miR-137表达水平是对照细胞的12.74倍。结论：成功包装miR-137过表达慢病毒，并可高效感染HEK293T细胞。

Packaging and identification of miR-137 overexpression lentivirus

Objective:To construct lentiviral vector which can overexpression miR-137 and produce lentivirus by lentivirus packaging system, and to explore its infection efficiency and expression in HEK293T cells.Methods: miR-137 sequence was chemically synthesized and cloned into lentiviral vector GV209, and the recombinant plasmid containing human miR-137 was obtained and identified.Then miR-137 recombinant plasmid together with two helper plasmids were transfected into HEK293T cells using Lipofectamine 2000.After the HEK293T cells were infected in multiplicity of infection(MOI) 40 for 48 h, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the expression level of miR-137 was detected by fluorescence quantitative PCR.Results:The sequencing results showed that the inserted gene sequence was completely consistent with the published human miR-137 gene sequence in GenBank.The GFP was observed in the HEK293T cells infected with miR-137 overexpression lentivirus under fluorescence microscope.The fluorescence quantitative PCR results showed that the expression level of miR-137 in the cells infected with overexpression lentivirus was 12.74 times higher than that in the control cells.Conclusion:The lentivirus containing miR-137 gene is successful packaged, and it could efficiently infect the HEK293T cells.