乙型肝炎病毒X基因异质性及对其反式激活功能的影响

EFFECT OF HEPATITIS B VIRUS X-GENE HETEROGENEITY ON ITS TRANSACTIVATION

从慢性乙型肝炎病毒 (HBV)感染患者外周血中扩增X基因序列 ,克隆入pGEM Teasy质粒 ,随机挑选克隆进行DNA测序以确定病毒的变异程度。结果提示 ,HBV长期携带者体内有HBV准种共存 ,X区内存在热点缺失突变区。为了进一步探讨X区突变对X蛋白反式激活功能的影响 ,将野生株及不同缺失突变的X基因片段克隆入pcDNA3 1( )载体的EcoRI位点 ,构建重组表达质粒 ,并分别与报告质粒pSV lacZ共转染HepG2细胞 ,提取细胞裂解液 ,检测SV40启动子调控下的 β 半乳糖苷酶表达活性。共转染结果显示 :野生株X蛋白能够反式激活SV40病毒早期启动子。

Polymerase chain reaction(PCR) was employed to amplify the whole HBV X region from the serum of patients with chronic hepatitis B virus(HBV) infection, and then the PCR products were subcloned into pGEM Teasy vectors. Clones were randomly selected to be sequenced. Comparison of the cloned sequence was made to look for the differene. The sequencing results showed that each sequence of selected clones was differenct and there were HBV quasispecies groups in patients. There was hot deletion region near the 3' end of X gene. To address whether the mutations were responsible for the transactivating effect,the wild type(wt) and the mutants of HBV X genes were subcloned into the EcoRI site of pcDNA3 1( ) vectors, and the recombinant plasmids were cotransfected into HepG2 cells with reporter plasmid pSV lacZ,respectively. The activity of β galactosidase controlled by SV40 early promoter/enhancer was detected, which reflected the transactivating function of HBx protein. The cotransfection results indicated that the wt HBV X gene acted as a transactivator on the SV40 early promoter/enhancer, and the mutations which caused premature termination of the X open reading frame reduced their transactivating effects to certain extent.