摘要
目的:研究HBV X基因与线粒体COXⅢ基因相互作用以及对线粒体功能的影响。方法:将HBV X基因真核表达载体pCDNA3-X及空载体pCDNA3转染成人肝细胞HL-7702细胞,经过2周G418筛选,获得稳定表达的新细胞株,分别命名为HL-7702/HBx和H-7702/pcDNA3,RT-PCR和Western blot方法证实HBV X基因在HL-7702/HBx细胞内的稳定表达。抽提细胞总RNA进行半定量RT-PCR及分离细胞线粒体进行Western blot检测COXⅢ表达水平变化,并通过酶动力学方法检测线粒体细胞色素氧化酶活性。结果:重组质粒pcDNA3-X转染后经G418筛选,RT-PCR和Western blot分析结果显示HL-7702-HBx细胞能够稳定表达HBV X基因。RT-PCR和Western blot结果发现HBV X基因下调COXⅢ蛋白表达而不影响mRNA水平表达。酶动力学检测发现线粒体细胞色素C氧化酶活性降低。结论:HBV X基因通过转录后水平调节COXⅢ表达,HBx通过与COXⅢ相互作用抑制线粒体细胞色素氧化酶活性。
AIM：To study the effect of HBx on a new HBx-interacted protein COXⅢ and mitochondria.METHODS：A recombinant eukaryotic expression plasmid pcDNA3-X was constructed,then pcDNA3-X and pcDNA3 were steadily transfected in HL-7702 separately,named HL-7702/HBx and HL-7702/pcDNA3.The expression of COXⅢ in HL-7702/HBx,HL-7702/pcDNA3 and HL-7702 cells were detected by RT-PCR and western blot.Mitochondrial cytochrome c oxidase activities of three groups were assayed spectrophotometrically at 550 nm by using partially purified mitochondria.RESULTS：COXⅢ mRNA expression didn＇t change,while protein expression is down-regulated in HL-7702/HBx.Cytochrome c oxidase activity was much lower in HL-7702/HBx compared with HL-7702 and HL-7702/pcDNA3 cells.CONCLUSION：HBx can down-regulate COXⅢ expression through post-transcriptional level and inhibit mitochondrial cytochrome c oxidase activity.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第10期972-974,978,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30770979)
福建省科技厅资助省属高校项目(2006F5043)
