摘要
本课题在体外转录翻译系统和稳定表达HBV的肝癌细胞中,研究La及其突变对HBVRNA的结合力、转录、翻译和病毒复制的影响。制备野生型La和突变体DeL1、DeL2、DeL3;构建adr HBV全基因、ORF-P、ORF-S、ORF-C和ORF-X真核表达质粒。应用电泳迁移率变动分析实验和体外转录翻译系统,研究La及其突变体对HBV RNA的迁移率、HBV及ORF转录和HBsAg、HBeAg表达的影响;在HepG2.2.15细胞内,采用SiRNA干扰细胞内La的表达,探讨La与HBV蛋白表达和复制的关系。结果发现:野生型La使HBV RNA迁移率显著降低且亲和力最强,不同位点的突变影响亲和结合力,RRM3缺失影响最显著;野生型La上调HBV全基因、P基因、C基因转录和HBsAg表达,抑制X基因转录和HBeAg表达,DeL2显著抑制X基因转录,DeL2和DeL3促进HBeAg表达;细胞内HBsAg、HBeAg和:HBV DNA含量与La的表达呈正相关。表明,RRM3与La的结合功能有关,RRM3和KRM2参与HBV转录和翻译启动的调控,La与HBV之间有密切相互作用关系,为寻找新的抗HBV药物靶点提供重要的实验依据。
The aim of this study was to investigate the effect of La and mutants on binding affinity, transcription, translation initiation of HBV RNA and HBV replication. Wild La, mutants and eukaryotic expression plasmid of adr HBV, ORF-P, ORF-S, ORF-C and ORF-X were constructed to research the influence of La or mutants on mobility of HBV RNA, HBV ORF transcription and HBsAg, HBeAg expression by EMSA and in vitro transcription-translation system. The relation of La and HBV replication was studied in HepG2.2.15 cell by RNAi. It suggested: the affinity of wild La and HBV RNA was perfect and different mutant sites influenced the binding affinity;the deletion of RRM3 had significant effect;wild La up-regulated HBV, ORF-P, ORF-C transcription and HBsAg expression, but it inhibited ORF-X transcription and HBeAg expression;DeL2 inhibited ORF-X transcription, but DeL2 and DeL3 promoted HBeAg expression notably;it was positive correlation between HBsAg, HBeAg, HBV DNA and La in cells. These results indicated: RRM3 was connected with the binding function;RRM3 and RRM2 were involved in transcription and translation initation regulation. It will be a novel antiviral strategies by studying the interaction of La and HBV.
引文
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