乙型肝炎病毒基因组C区启动子肝富集转录因子结合位点突变对HBx增强乙型肝炎病毒复制的影响

Role of the liver-enriched transcription factor binding site mutation in the C promoter region of hepatitis B virus genome for HBx-enhanced hepatitis B virus replication

目的构建乙型肝炎病毒(HBV)C启动子近端肝富集转录因子结合位点突变的重组HBV复制型质粒,以期阐明其在HBx增强HBV复制中的作用。方法利用定点突变技术在野生型HBV复制型质粒和HBx表达缺失的HBV复制型质粒基础上构建HBV C启动子近端肝富集转录因子结合位点突变的重组质粒。随后在HBV肝癌细胞复制模型和小鼠复制模型中进行质粒转染,提取细胞和小鼠肝组织的HBV复制中间体进行检测。结果在HBV复制型质粒的基础上成功构建HBV C启动子近端肝富集转录因子结合位点突变的HBV复制型质粒。在HBV肝癌复制模型和小鼠复制模型中均发现HBx增强HBV的复制。突变HBV C启动子肝富集转录因子结合位点以后,HBx对于HBV复制增强的作用并未受到显著影响。结论HBx增强HBV复制可能不通过肝富集转录因子和与HBV C启动子近端的结合位点而起作用,其他肝富集转录因子结合位点在HBx增强HBV复制中的作用仍需进一步研究。

Objective To construct a recombinant HBV replication-type plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter in order to elucidate the role of HBx-enhanced HBV replication.Methods Site-directed mutagenesis technology was used to construct a recombinant plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter on the basis of wild-type HBV replicating plasmid and HBV replicating plasmid lacking HBx expression.Subsequently,plasmid transfection was carried out in HBV liver cancer cell replication model and mouse replication model,and HBV replication intermediates of cells and mouse liver tissue were extracted for detection.Results Based on the HBV replicating plasmid,the HBV replicating plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter was successfully constructed.HBx-enhanced HBV replication were detected in both the HBV liver cancer replication model and the mouse replication model.After mutating liver-enriched transcription factor binding site mutation at proximal of HBV C promoter,the effect of HBx on the enhancement of HBV replication was not significantly affected.Conclusion HBx may not enhance HBV replication through liver-enriched transcription factor binding site mutation at proximal of HBV C promoter.The role of other liver-enriched transcription factor binding sites in HBx-enhanced HBV replication needs further study.