桑圣刚,王梦旖,肖 曼,杜冠魁.乙型肝炎病毒HBx基因的shRNA重组表达载体的构建和鉴定[J].中南民族大学学报自然科学版,2018,(4):55-60
乙型肝炎病毒HBx基因的shRNA重组表达载体的构建和鉴定
Construction and Identification of shRNA RecombinantExpression Vector for HBx Gene of Hepatitis B Virus
  
DOI:10.12130/znmdzk.20180412
中文关键词: 乙型肝炎病毒  HBx 基因  RNA 干扰,基因表达
英文关键词: hepatitis B virus  HBx gene  RNA interference, gene expression
基金项目:国家自然科学基金资助项目( 81360244)
作者单位
桑圣刚,王梦旖*,肖 曼,杜冠魁 海南医学院 基础医学院海口 571100 
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中文摘要:
      目的: 构建 pGenesil-3.1-HBx-shRNA 重组表达载体, 探讨其作用于 HepG2.2.15 细胞的生物活性. 方法: 构建了针对HBx基因的siRNA 表达载体,通过电穿孔法转染 HepG2.2.15 细胞,以荧光显微镜观察细胞的转染效率,QRT-PCR检测细胞中HBx基因的相对表达量,利用CCK-8细胞增殖检测试剂盒检测了细胞增殖活力,流式细胞术检测细胞凋亡.结果: 酶切和测序鉴定证实构建质粒含有HBx基因,QRT-PCR检测和荧光显微镜观察到构建的pGenesil-3.1-HBx-shRNA 可以在 HepG2.2.15 细胞中表达,HBx基因的表达下降了 28%(P<0.05),细胞增殖水平下降了47%(P<0.05),质粒 pGenesil-3.1-HBx-shRNA 转染后的HepG2.2.15 细胞凋亡率显著升高.结论: 乙型肝炎病毒HBx基因的shRNA重组表达载体构建成功,沉默HBx基因的shRNA能够抑制HBx基因的表达,且对HepG2.2.15细胞的增殖水平起明显的抑制作用.
英文摘要:
      Objective: To construct a recombinant expression vector of pGenesil-3.1-HBx-shRNA and investigate its antihepatitis B virus (HBV) effect. Methods: The siRNA expression vector targeting HBx gene was designed and constructed.HepG2.2.15 cells were transfected by electroporation. The transfection efficiency of the cells was observed by fluorescence microscopy.The relative expression of HBx gene in the cells was detected by QRT-PCR.The cell proliferation assay kit was used to detect cell proliferation activity. The effects of apoptosis were detected by the Annexin V Apoptosis Kit.Results:The recombinant plasmid containing HBx gene was confirmed by the restriction enzyme digestion and sequencing. The pGenesil-3.1-HBx-shRNA could be expressed in HepG2.2.15 cells by QRT-PCR and fluorescence microscopy,and the expression of HBx gene was decreased by 28%(P<0.05),the cell proliferation level decreased by 47%(P<0.05),and the apoptosis rate of HepG2.2.15 cells transfected with plasmid pGenesil-3.1-P-shRNA was significantly increased.Conclusion: The shRNA recombinant expression vector of hepatitis B virus HBx gene was successfully constructed. The shRNA silencing HBx gene could inhibit expression of theHBx gene,and inhibit the proliferation of HepG2.2.15 cells.
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