核定位信号突变型P21真核表达载体的构建及其对HepG2.2.15细胞病毒复制的影响

目的构建核定位信号突变型P21基因的真核表达载体并初步探讨其对HBV复制的影响。方法采用基因定点诱变技术突变P21基因的核定位信号序列,采用DNA重组技术,亚克隆突变型P21基因至pDsRed1-C1真核表达载体中,重组为pDsRed1-C1-p21NLS-,酶切鉴定及DNA测序,脂质体转染HepG2.2.15细胞,RT-PCR检测目的基因pDsRed1-C1-p21NLS-的表达,荧光显微镜观测目的基因亚细胞定位,ELISA法检测培养上清液中HBsAg、HBeAg的水平。结果 pDsRed1-C1-p21NLS-质粒构建成功,与野生型相比,主要在胞浆表达,促进病毒的复制。二者差异有统计学意义（P〈0.01）。结论 P21的亚细胞定位,对HepG2.2.15细胞中病毒复制的影响具有明显差异。胞核P21抑制病毒的复制,而胞浆P21促进病毒复制。

Construction of eukaryotic expression vector of P21 mutation type by nuclear location signal and its effect on virus replication in HepG2.2.15 Cells

Objective To construct the eukaryotic expression vector of pDsRed1-C1-p21NLS-gene and study its function.Methods The mutation with nuclear localization signal of p21 was obtained by site-directed mutagenesis.p21 NLS-was directly cloned into eukaryotic expressive plasmid pDsRed1-C1 and transfected into HepG2 Cells by liposome-mediated DNA transfection.The p21 NLS-gene expression,intracellular localization of the p21 and HBV antigens were detected by RT-PCR,fluorescence microscope and ELISA,respectively.Results The expression of pDsRed1-C1-p21NLS-was mainly localized in the cytoplasm compared to pDsRed1-C1-p21WT which was localized in the nucleus.The expression of pDsRed1-C1-p21NLS-improved the replication of HBV in HepG2.2.15.Conclusion The different intracellular localization of p21 has various impacts on replication of HBV in HepG2.2.15 cells.