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目的为了探讨乙型肝炎病毒(HBV)X基因与慢性乙型肝炎及肝癌发生的关系,构建HBV X基因(hep-atitis B virus X gene,HBx)真核表达载体及其肝癌(HCC)细胞系HepG2瞬转模型,并检测HBx对HepG2细胞表型的影响。方法以pCMV-HBx为模板,PCR法扩增HBx基因编码区全长序列,将其克隆至pcDNA3.1-Flag真核表达载体中,构建重组真核表达载体pcDNA3.1-Flag-HBx,转染至HepG2细胞;Western印迹法检测细胞中HBx蛋白的表达水平;5-溴尿苷(5-溴脱氧尿嘧啶核苷,5-bromo-2’-deoxyuridine,BrdU)标记法检测转染HBx后细胞DNA合成变化;细胞周期检测试剂盒(CycleTESTTMPLUS DNA Reagent Kit)检测转染HBx后细胞周期的变化;AnnexinⅤ-APC/7-AAD法检测转染HBx后细胞凋亡情况。结果重组真核表达载体pcDNA3.1-Flag-HBx经双酶切和测序证明构建正确,转染该载体的HepG2细胞可检测到HBx蛋白的表达;与转染空质粒pcDNA3.1-Flag的细胞相比,细胞增殖能力增强,而细胞凋亡比率下降。结论成功构建了HBx基因真核表达载体,并研究了HBx对细胞表型的影响,为进一步探索HBx参与HBV引发HCC的分子机制奠定了基础。
Objective To investigate the relationship between hepatitis B virus (HBV) X gene and the occurrence of chronic hepatitis B and hepatocellular carcinoma (HCC) and to construct the eukaryotic expression vector of HBV X gene (HBx) and its hepatocellular carcinoma (HCC) cell line HepG2 transient model and detect the effect of HBx on the phenotype of HepG2 cells. Methods The full-length coding sequence of HBx gene was amplified by PCR using pCMV-HBx as a template and cloned into pcDNA3.1-Flag eukaryotic expression vector to construct recombinant eukaryotic expression vector pcDNA3.1-Flag-HBx Stained to HepG2 cells; Western blotting was used to detect the expression of HBx protein; 5-bromo-2’-deoxyuridine (BrdU) The changes of cell DNA synthesis were detected by MTT assay. The cell cycle was detected by CycleTEST TMPLUS DNA Reagent Kit. The cell apoptosis was detected by AnnexinⅤ-APC / 7-AAD assay. Results The recombinant eukaryotic expression vector pcDNA3.1-Flag-HBx was confirmed by double enzyme digestion and sequencing. The expression of HBx protein was detected in HepG2 cells transfected with the vector. Compared with the cells transfected with empty plasmid pcDNA3.1-Flag In contrast, cell proliferation increased, while the rate of apoptosis decreased. Conclusion The HBx gene eukaryotic expression vector was successfully constructed and the effect of HBx on cell phenotype was studied. This study laid the foundation for further exploration of the molecular mechanism of HBx participating in HBV-induced HCC.