HCV核心蛋白与HBV X蛋白协同反式激活作用的研究

Synergetic transactivating effect of HCV core and HBV X proteins on SV40 early promoter/enhancer

目的 探讨HCV核心蛋白与HBVX蛋白的协同反式激活作用。方法 构建表达HCV核心蛋白的重组质粒pcDNA3 .1( )core和表达HBVX蛋白的重组质粒pcDNA3 .1( )X ;转染HepG2 细胞 ,从转录和翻译水平鉴定病毒基因的瞬时表达 ;与报告质粒pSV lacZ共转染HepG2 细胞 ,检测 β 半乳糖苷酶表达活性 ,酶的活性反映了表达的肝炎病毒蛋白对SV40病毒早期启动子 增强子功能的影响。结果 构建成HCV核心蛋白及HBVX蛋白的重组表达载体pcDNA3 .1( )core、pcDNA3 .1( )X ;在HepG2 细胞均能瞬时表达相应的肝炎病毒蛋白 ;单独共转染试验中pcDNA3 .1( )core、pcDNA3 .1( )X组的 β 半乳糖苷酶的表达分别是对照的 4.9、3 .5倍 ,两种质粒共同转染时酶的表达是对照的 9倍 ;表达质粒对 β 半乳糖苷酶表达的激活作用呈剂量依赖性。 结论 HepG2 细胞中表达的HCV核心蛋白和HBVX蛋白均具有反式激活SV40早期启动子 增强子的功能 ,并且两种蛋白的反式激活功能具有协同特性。本试验有助于解释HCV、HBV感染 ,尤其是共同感染的致病

Objective To investigate the synergetic transactivating effects of HCV core and HBV X proteins. Methods HCV core and HBV X protein expressing plasmids were constructed with the vector pcDNA3 1( ). The plasmids were transfected into HepG 2 cells and cotransfected HepG 2 cells with reporter plasmid pSV lacZ by lipofectamine plus reagents. The virus proteins produced in transient expression system were detected at the transcription and translation levels. The activity of β galactosidase was detected, which reflected the transactivating function of the proteins. Results The expression of plasmids were detected in soluble protein cell extracts of transiently transfected HepG 2 cells. HCV core protein activated the β galactosidase expression at a value 4.9 times higher than the control, while HBV X protein activated at a value 3.5 times. It arrived at 9 times transfected with the plasmids simultaneously. The activating effect increased in relation to the amount of plasmids. Conclusion The results suggested that the two kinds of virus proteins have transactivating effect on SV40 early promoter/enhancer, and they acted synergistically. These contribute to explain the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection.