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目的构建并鉴定靶向人转录因子FOXM1基因的shRNA慢病毒载体。方法设计合成靶向人FOXM1基因的shRNA序列,通过基因重组技术将其连接到含U6启动子及绿色荧光蛋白基因的慢病毒表达载体pLL3.7中,构建shRNA重组质粒pLL-FOXM1-shRNA。重组质粒经XbaⅠ和NheⅠ双酶切及DNA测序鉴定后,与慢病毒包装质粒混合,与脂质体Lipofec-tamineTM 2000共转染至293T细胞中,孵育72 h后收集上清液,高速离心纯化病毒颗粒,并采用逐孔稀释法测定慢病毒滴度。结果双酶切鉴定及测序结果证实,靶向人FOXM1基因的shRNA序列已成功插入pLL3.7中;在293T细胞中成功包装出慢病毒颗粒,病毒滴度为1×108 TU/ml。结论 已成功构建了靶向人FOXM1基因的shRNA慢病毒载体,为进一步研究FOXM1的分子功能奠定了基础。
Objective To construct and identify shRNA lentivirus vector targeting human transcription factor FOXM1. Methods shRNA targeting human FOXM1 gene was designed and synthesized. The recombinant plasmid pLL-FOXM1-shRNA was constructed by ligating it to lentiviral vector pLL3.7 containing U6 promoter and green fluorescent protein gene by gene recombination technology. Recombinant plasmids were digested with XbaⅠ and NheⅠ and identified by DNA sequencing. The recombinant plasmids were mixed with the lentivirus packaging plasmid and co-transfected into 293T cells with Lipofectamine 2000. After 72 hours of incubation, the supernatants were collected and purified by high speed centrifugation The virus particles and by dilution method to measure lentivirus titer. Results Double enzyme digestion and sequencing confirmed that the shRNA sequence targeting FOXM1 gene was successfully inserted into pLL3.7. Lentiviral particles were successfully packaged in 293T cells and the virus titer was 1 × 108 TU / ml. Conclusion The shRNA lentiviral vector targeting human FOXM1 gene has been successfully constructed, which lays the foundation for further research on the molecular function of FOXM1.