B、C基因型乙型肝炎病毒X蛋白结构对反式激活能力影响

Difference in structural and transactivating capacity between X proteins of genotype B and C hepatitis B virus

目的 研究B、C基因型乙型肝炎病毒 (HBV)X蛋白氨基酸差异对反式激活能力的影响。方法 以DNAMAN软件对GenBank中B、C基因型HBV的X蛋白进行氨基酸同源比较 ,确定B、C基因型HBVX蛋白各自的保守氨基酸序列并分析基因型特异性氨基酸。以相应核苷酸序列为参照 ,通过基因定点突变的方式获得保守的B、C型HBVX基因 (分别为XB及XC)并构建真核表达载体pcDNA3.1 XB及pcDNA3.1 XC。将表达载体与报告质粒pCMVβ(含CMV即刻早期启动子 )共同转染HepG2细胞并检测细胞内 β 半乳糖苷酶的活性 ,实验数据以配对t检验进行分析。结果 B、C型HBVX蛋白之间共有 17个氨基酸差异 ,主要位于蛋白的反式抑制区及反式激活功能区。β 半乳糖苷酶在各组转染细胞内的活性依次为pcDNA3.1 XB +pCMVβ组 >pcDNA3.1 XC +pCMVβ组 >pcDNA3.1 Hygro( ) +pCMVβ组 (对照组 )。结论 B、C基因型HBVX蛋白对CMV即刻早期启动子均有反式激活能力 ,且B基因型HBVX蛋白要强于C基因型 ,这种差别与B、C基因型间X蛋白的 17个氨基酸差异有关。X蛋白反式激活能力的不同与B、C基因型HBV产生不同的临床及病理表现的关系有待进一步研究。

Objective To compare the structural and transactivating capacity of genotype B and C HBV X proteins. Methods By amino acids alignment using DNAMAN software,we identified the consensus amino acids of genotype B and C HBV X protein separately and examined B or C genotype-specific amino acids of X proteins. Obtained the genotype B and C HBV X gene with consensus sequences (namely,XB or XC) by means of site-directed mutagenesis,and constructed the recombinant eukaryotic expression vectors pcDNA3.1-XB and pcDNA3.1-XC. Co-transfected the recombinant vector with reporter plasmid pCMVβ (harboring CMV immediate-early promoter) to HepG2 cells and monitored the intracellular β-galactosidase activities of transfected cells. Results There were 17 amino acids differences between genotype B and C HBV X proteins and most of them were located in the transrepression and transactive domains. Among three groups of transfected HepG2 cells,intracellular β-galactosidase activities reduced accordingly as pcDNA3.1-XB+pCMVβ>pcDNA3.1-XC+pCMVβ>pcDNA3.1/Hygro(-)+pCMVβ. Conclusion Both the genotype B and C HBV X protein could transactivate the CMV immediate-early promoter,the transactivating capacity of genotype B X protein was higher than that of genotype C. The different transactivating capacities between genotype B and C HBV X protein were explained by 17 amino acids variation and the relationship of X protein differentiations with clinical manifestations and pathological profiles of genotype B and C HBV infection was needed to be elucidated.