靶向人转录因子FOXM1基因的shRNA慢病毒载体的构建与鉴定

Construction and Identification of Lentivirus-mediated Short Hairpin RNA Targeting Human FOXM1 Gene

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 a lentivirus-mediated short hairpin RNA（shRNA） targeting human FOXM1 gene.Methods The shRNA sequence targeting to human FOXM1 gene was designed and synthesized,then linked to lentivirus expression vector pLL3.7 containing U6 promoter and green fluorescent protein（GFP） gene by gene recombination technique.The constructed recombinant plasmid pLL-FOXM1-shRNA was identified by digestion with XbaⅠand NheⅠand DNA sequencing,then mixed with lentivirus packaging plasmids and co-transfected to 293T cells with LipofectamineTM 2000.Supernatant was collected 72 h after incubation,from which lentivirus particles were purified by high speed centrifugation and determined for titer by hole-by-hole dilution method.Results Both restriction analysis and sequencing proved that shRNA sequence targeting human FOXM1 gene was successfully inserted into vector pLL3.7.Lentivirus particles were successfully packaged in 293T cells,of which the titer was 1 × 108 TU / ml.Conclusion The lentivirus-mediated shRNA targeting human FOXM1 gene was successfully constructed,which laid a foundation of further study on molecular function of FOXM1.