丙型肝炎病毒非结构蛋白NS3与乙型肝炎病毒 X蛋白协同反式激活作用的研究

构建丙型肝炎病毒（HCV）非结构蛋白3（NS3）的重组表达质粒pcDNA3.1(－）-NS3和表达乙型肝炎病毒（HBV）X蛋白的重组质粒pcDNA3.1(－）-X，分别瞬时转染肝母细胞瘤细胞系HepG2细胞，用免疫印记（Western blotting)方法鉴定病毒基因的表达。两个表达质粒分别及同时与报告质粒pCAT 3-promoter共转染HepG2细胞，检测报告基因氯霉素乙酰转移酶（CAT）的表达水平，酶的活性反映了表达的肝炎病毒蛋白对SV40病毒早期启动子功能的影响。结果显示，两个重组表达载体pcDNA3.1(－）-NS3及pcDNA3.1（－）-X在HepG2细胞均以瞬时表达相应的肝炎病毒蛋白；单独共转染实验中pcDNA3.1(－）-NS3.1(－）-X组的CAT的表达分别是对照的3．6倍和4．4倍，两种质粒共同转染时酶的表达是对照的8．5倍；表达质粒对CAT酶表达的激活作用呈剂量依赖性。研究表明，HepG2细胞中表达的HCV NS3蛋白和HBV X蛋白均具有反式激活SV40早期启动子的功能，并且两种蛋白的反式激活功能具有协同特性。本实验有助于解释HCV、HBV感染，尤其是共同感染的致病（癌）机制。

STUDY OF SYNERGETIC TRANSACTIVATING EFFECT OF HCV NS3 AND HBV X PROTEINS ON SV40 EARLY PROMOTER

Hepatitis C virus (HCV) non structure 3 (NS3) protein and hepatitis B virus (HBV) X protein expressing plasmids were constructed with the vector pcDNA3 1(-). The plasmids were transfected into HepG2 cells and the viral proteins expressed in HepG2 cells were identified using Western blotting methods. Then the two recombined plasmids were transfected into HepG2 cells and were cotransfected into HepG2 cells with reporter plasmid pCAT3 promoter. The activity of CAT enzyme was detected by a CAT ELISA assay kit, which reflected the transactivating function of two proteins on SV40 early promoter. The findings indicated that the expression of two viral proteins were successfully detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV NS3 protein transactivated the CAT enzyme expressed at a value 3 5 fold higher than the control, while HBX protein transactivated at a value 4 4 times. It arrived at 8 5 times when transfected with two plamids simultaneously. The activating effect was increased in relation to the amount of plasmids. It was suggested that the two kinds of viral proteins had a transactivating effect on SV40 early promoter, and they acted synergistically. These results might contribute to explaining the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection