hsa-miR-30e的克隆及其慢病毒表达载体的构建

Construction of Recombinant Lentiviral Vector Expressing hsa-miR-30e

目的克隆hsa-miR-30e并构建其慢病毒表达载体。方法 PCR扩增hsa-miR-30e的前体序列,克隆于慢病毒载体pll3.7,转染包装细胞293FT细胞,收获病毒颗粒,荧光定量PCR检测hsa-miR-30e的表达。结果经双酶切及测序鉴定,hsa-miR-30e的慢病毒表达载体构建成功,倒置荧光显微镜下观察转染后293FT细胞发出绿色荧光。结论成功构建hsa-miR-30e的慢病毒表达载体,为进一步研究hsa-miR-30e的生物学功能奠定了基础。

Objective To construct a lentiviral vector expressing microRNA, hsa-miR-30e. Methods Pri- miR-30e amplified by PCR was inserted into pt13.7 vector,and then confirmed by restriction endonuclease analysis and DNA sequencing.Then 293FT cells were contransfected with recombinant plasmid and helper plasmid.The expression of miR-30e was detected by fluorescence and Real-time PCR. Result The recombinant plasmid contains the target sequence of miR-30e and can express GFP successfully. Conclusion The constructed lentiviral vector can express miR-30e in vitro, providing the fundation for further study of miR-30e.