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

邱荣元; 何生松; 陈锋; 庞然

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

1. 华中科技大学同济医学院附属协和医院肝病科
2. 岳阳市第二人民医院消化内科

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中图分类号: R512.62
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出版历程
- 出版日期: 2010-04-20

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

摘要

摘要: 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。
- 核定位信号 /
- 肝炎病毒,乙型 /
- 病毒复制 /
- HepG2.2.15细胞
Abstract: 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.
- nuclear localizaion signals /
- hepatitis B virus /
- virus replication /
- HepG2.2.15 cell

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