miR-186过表达慢病毒载体的构建及鉴定

目的：构建miR-186过表达慢病毒载体并包装慢病毒，探讨miR-186在人胚胎肾细胞（HEK）293T细胞系中的感染效率和表达水平。方法：以Hsa-miR-186前体序列为模板，设计并合成引物，PCR法扩增pre-miR-186基因序列，并将其克隆到携带EGFP/Puromycin的慢病毒载体FV040中，经EcoRⅠ和AgeⅠ酶切及测序鉴定后获得重组慢病毒载体。利用Lipofectamine 2000将重组慢病毒质粒FV040 Vector和FV040 miR-186分别与辅助质粒通过共转染至HEK293T细胞中，48 h后收集慢病毒，以FV040 Vector慢病毒作为对照组，FV040 miR-186作为实验组，分别感染HEK293T细胞。感染48 h后，观察HEK293T细胞中绿色荧光的分布情况，并采用实时荧光定量PCR法检测miR-186的表达水平。结果：测序分析，miR-186过表达慢病毒与GenBank上公布的miR-186序列完全一致。与对照组（0.8387±0.1456）比较，实验组HEK 293T细胞中miR-186表达水平（12.6400±0.7884）明显升高（t=14.72，P<0.01），约为对照组的15.07倍。结论：成功构建miR-186过表达慢病毒载体并包装出慢病毒，miR-186慢病毒成功感染HEK293T细胞，miR-186表达水平在HEK293T细胞中明显升高。

Construction and identification of miR-186 overexpression lentiviral vector

Objective:To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and toexplore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods:The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoRⅠand AgeⅠrestriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results:The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×10⁸ TU·mL^-1 and 5×10⁸ TU·mL^-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12.640 0±0.788 4) was significantly increased by 15.07 times (t=14.72,P<0.01) compared with control group (0.838 7±0.145 6). Conclusion:The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus suceessfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.