乙型肝炎病毒x蛋白基因荧光真核表达质粒的构建及其对人正常肝细胞株L0_2增殖的影响

Construction of fluorescent eukaryotic expression vector for hepatitis B virus x protein and its effect on human normal liver cell strain L0_2

目的构建乙型肝炎病毒x蛋白基因荧光真核表达质粒,并检测HBx对人正常肝细胞株L02细胞增殖的影响。方法以pcDNA3-HBV质粒为模板,PCR法扩增HBx基因编码区全长序列,将其克隆至pIRES2-EGFP荧光真核表达载体中,构建重组真核表达质粒pIRES2-EGFP-HBx,转染L02细胞,筛选稳定表达HBx的细胞,RT-PCR法检测细胞中HBx基因mRNA的转录水平;Western blot法检测细胞中HBx蛋白的表达水平;MTT法检测细胞的增殖活力。结果重组荧光真核表达质粒pIRES2-EGFP-HBx经双酶切和测序证明构建正确,转染该质粒的L02细胞可检测到HBx基因mRNA的转录及蛋白的表达,与空质粒pIRES2-EGFP转染的L02细胞相比,增殖活力明显提高。结论成功构建了HBx基因荧光真核表达质粒,并获得了能稳定表达HBx蛋白的L02细胞株,为进一步研究HBx对细胞周期调控通路的影响及探索HBx导致的与HBV相关的HCC的分子机制奠定了基础。

Objective To construct a fluorescent eukaryotic expression vector for hepatitis B virus （HBV） x protein and determine its effect on proliferation of human normal liver cell strain L02. Methods Full-length sequence at encoding region of HBx gene was amplified by PCR using plasmid pcDNA3-HBV as a template, and cloned into fluorescent expression vector plRES2-EGFP. The constructed recombinant plasmid plRES2-EGFP-HBx was tranfected to L02 cells, based on which the cells stably expressing HBx were screened, and determined for transcription level of HBx mRNA by RT-PCR, for expression level of HBx protein by Western blot, and for proliferation activity by MTF method. Results Restriction analysis and sequencing proved that recombinant fluorescent eukaryotic expression vector plRES2-EGFP-HBx was constructed correctly. Both the transcription of HBx mRNA and expression of HBx protein were proved in the L02 cells transfected with plasmid plRES2-EGFP-HBx. The proliferation activity of L02 cells transfected with plasmid plRES2-EGFP-HBx was significantly higher than that with empty vector plRES2-EGFP. Conclusion The fluorescent eukaryotic expression vector for HBV x protein was successfully constructed, and L02 eeU strain stably expressing HBx was screened, which laid a foundation of further study on effect of HBx on regulatory pathway of cell cycle as well as the molecular mechanism of HBV-associated HCC caused by HBx.