HBx基因的真核表达及其对肝癌细胞系HepG2细胞表型的影响

The eukaryotic expression of hepatitis B virus X gene and its effect on the cellular phenotype of HepG2 cell line

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 construct a eukaryotic expression vector for hepatitis B virus （HBV） X protein and determine its effect on proliferation of hepatic carcinoma cell strain HepG2. Methods A full-length sequence of HBx ORF was ampli- fied by PCR using plasmid（pCMV-HBx） as a template, and cloned into eukaryotic expression vector pcDNA3.1-Flag. The constructed recombinant plasmid pcDNA3. 1-Flag-HBx was transfected to HepG2 cells. The transcription level of HBx mRNA by RT-PCR and expression level of HBx protein were deternined by Western blot. The cell proliferation rate via the 5-bromo-2＇-deoxyuridine（BrdU） and propidium iodide assay,while cell apoptosis by Annexin V-APC/7-AAD method. Results Restriction analysis and sequencing proved that recombinant eukaryotic expression vector pcDNA3.1-Flag-HBx was constructed correctly. The expression of HBx protein was proved in the HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx. The cell proliferation ability of HepG2 cells transfected with plasmid pcDNA3. 1-Flag-HBx was increased compared with empty vector pcDNA3.1-Flag. Meanwhile, the cell apoptosis ratio of HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx was lower than that with empty vector pcDNA3.1-Flag. Conclusion The eukaryotic expres- sion vector for HBV X protein is successfully constructed, and the cell proliferation ability of HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx is increased compared with empty vector, while the cell apoptosis ratio of HepG2 cells trans- letted with plasmid pcDNA3.1-Flag-HBx is lower than that with empty vector pcDNA3. 1-Flag. This research will help study the effect of HBx on the regulatory pathway of cell phenotype as well as the molecular mechanism of HBV-associated hepatocellular carcinoma（HCC） caused by HBx.