靶向定位的乙型肝炎病毒X蛋白真核表达载体构建及表达  

Construction of HBV X protein expression vectors and detection of localized HBx nuclear or mitochondria expression

作　　者：肖珑 胡瑾华 段学章 刘晓燕 刘士敬 荣义辉 辛绍杰 

机构地区：[1]解放军第三Ο二医院肝衰竭诊疗与研究中心,北京100039 [2]解放军第三Ο二医院中西医结合肝病诊疗与研究中心,北京100039

出　　处：《中国病毒病杂志》2012年第6期455-459,共5页Chinese Journal of Viral Diseases

摘　　要：目的构建带有核定位信号(NLS)和线粒体定位信号(MLS)的乙型肝炎病毒X蛋白(HBx)真核表达载体,并检测其在人胚肾293FT细胞中的表达和亚细胞定位情况。方法采用PCR技术分别扩增出增强型绿色荧光蛋白(EGFP)和HBx序列,人工合成编码NLS和MLS的核酸序列,依次将其克隆至pcDNA4.0载体中。将重组质粒转染293FT细胞,Western blot检测融合蛋白的表达情况,荧光显微镜观察带有定位标签的HBx在细胞中的定位。结果测序鉴定结果表明带有NLS、MLS和EGFP标签的HBx真核表达载体构建成功,转染293FT细胞能够正常表达融合蛋白,在荧光显微镜下,NLS和MLS能够将EGFP-HBx融合蛋白分别定位于细胞核和线粒体。结论本研究构建的能够特异性靶向HBx蛋白至细胞核和线粒体的真核表达载体,能够在哺乳动物细胞293FT细胞中表达,为进一步深入研究不同定位HBx的生物学功能奠定了基础。Objective To construct the eukaryotic expression vector encoding hepatitis B virus X（HBx）protein carried with both nuclear and mitochondria localization signals,and to detect its expression,subcellular location in human kidney 293FT cells.Methods The EGFP and HBx DNA sequences were obtained by PCR amplification and cloned into pcDNA4.0 plasmid together with either synthesized NLS or MLS nucleotide sequences.Human 293FT cells were transduced with the recombinant plasmids pcDNA4.0-MLS-EGFP-HBx and pcDNA4.0-NLS-EGFP-HBx;the expression and subcellular location of HBx fusion proteins were detected by Western blot and fluorescence microscope.Results HBx eukaryotic expression vectors fused with MLS and NLS and EGFP were successfully constructed and the expressions of HBx fusion proteins were verified by Western blot.Fluorescence microscope analysis showed that the EGFP-HBx fusion proteins could be targeted into either nuclear or mitochondria by NLS and MLS,respectively.Conclusions The constructed eukaryotic expression vectors MLS-EGFP-HBx and NLS-EGFP-HBx successfully expressed fusion proteins in the nuclear and mitochondria,respectively.

关 键 词：乙型肝炎病毒X蛋白 核定位信号 线粒体定位信号 亚细胞定位 

分 类 号：R512.62[医药卫生—内科学]