人甘油激酶慢病毒干涉载体的构建和表达

Construction of a lentiviral vector for RNA interference of glycerol kinase gene in human hepatocytes in vitro

目的构建人甘油激酶(GK)基因RNA干扰(RNAi)慢病毒载体,获得稳定下调GK表达的慢病毒。方法将具有干涉效果的siRNA序列克隆入慢病毒核心质粒pSicoR中,并转染293T细胞进行病毒包装,用慢病毒感染293T细胞并应用FACS测定病毒滴度。以GK干涉慢病毒感染人肝细胞系L02细胞后,应用Western blotting方法检测GK的表达。结果成功构建GK干涉慢病毒pSicoR-GK载体并获得慢病毒颗粒,病毒滴度检测可达3×107pfu/ml,GK蛋白质表达水平下调至对照的约20%。结论成功构建人GK基因RNAi慢病毒,为后续GK生物学功能研究奠定基础。

Objective To construct a lentiviral vector for RNA interference（RNAi） of human glycerol kinase（GK） gene to stably down-regulate GK expression in human hepatocytes.Methods The sequence of siRNA for GK interference were cloned into the pSicoR vector.Following packaging in 293T cells,the lentivirus was titrated using fluorescence activated cell sorting.Human hepatocyte L02 cells was infected with the lentivirus and the expression of GK was analyzed using Western blotting.Results The lentiviral particle pSicoR-GK was successfully packaged with a virus titer reaching 3×107 pfu/ml.The expression level of GK protein was down-regulated to 20% of the control level in L02 cells infected with the lentivirus.Conclusion The lentiviral vector for RNAi of human GK gene has been successfully constructed,which can significantly down-regulate GK expression in human hepatocytes.