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目的克隆微小RNA hsa-miR-106b并构建其慢病毒表达载体。方法将PCR扩增得到的miR-106b前体序列和pLVTHM载体经双酶切后连接,产生pLVTHM-miR-106b慢病毒表达载体,双酶切后测序鉴定,筛选阳性克隆。用pLVTHM-miR-106b、psPAX2和pMD2.G质粒共转染包装细胞293FT,包装产生慢病毒。结果经双酶切鉴定和测序证实,成功构了miR-106b的慢病毒表达载体pLVTHM-miR-106b。倒置荧光显微镜下观察可见包装细胞293FT呈绿色荧光。结论成功构建了has-miR-106b的慢病毒表达载体,为深入研究miR-106b的生物学功能奠定基础。
Objective To clone hsa-miR-106b and construct its lentiviral vector. Methods The amplified product of miR-106b and pLVTHM vector were double-digested and ligated to generate the lentiviral vector pLVTHM-miR-106b. After double digestion and sequencing, the positive clones were screened. 293FT packaging cells were co-transfected with pLVTHM-miR-106b, psPAX2 and pMD2.G plasmids to produce lentivirus. Results The double-restriction enzyme digestion and sequencing confirmed that miR-106b lentiviral vector pLVTHM-miR-106b was successfully constructed. Inverted fluorescence microscopy showed that the packed cells 293FT was green fluorescence. Conclusion The lentiviral expression vector with has-miR-106b has been successfully constructed, which lays the foundation for further study on the biological function of miR-106b.