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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 plasmid of hepatitis B virus X gene and detect the effect of HBx on the proliferation of human normal liver cell line L02. Methods The full-length coding sequence of HBx gene was amplified by PCR using pcDNA3-HBV plasmid as a template. The full-length sequence of HBx gene was amplified by PCR and cloned into pIRES2-EGFP fluorescent eukaryotic expression vector. The recombinant eukaryotic expression vector pIRES2-EGFP-HBx was constructed and transfected into L02 Cells were screened for stable expression of HBx cells. RT-PCR was used to detect the transcription level of HBx mRNA in the cells. Western blot was used to detect the expression of HBx protein. Cell viability was measured by MTT assay. Results The recombinant plasmid pIRES2-EGFP-HBx was confirmed by double enzyme digestion and sequencing. The transfection and expression of HBx gene mRNA were detected in L02 cells transfected with pIRES2-EGFP, Compared with L02 cells, the proliferative activity was significantly increased. Conclusion The HBx gene eukaryotic expression plasmid was successfully constructed and the L02 cell line stably expressing HBx protein was obtained. To further investigate the effect of HBx on the cell cycle regulatory pathway and to explore the molecular mechanism of HBV-associated HCC induced by HBx The foundation.