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为研究NIRF(Np95/ICBP-90 like RING finger protein)对乙型肝炎病毒(hepatitis B virus,HBV)的复制以及与乙型肝炎病毒共价环状闭合DNA(HBV cccDNA)结合的组蛋白H3乙酰化的影响,采用脂质体转染将pGEM-HBV1.3+pFLAG、pGEM-HBV1.3+pFLAG-NIRF、pGEM-HBV1.3质粒分别转入HepG2细胞,Western blot检测NIRF蛋白的表达,用ELISA结合RT-PCR检测HBsAg、HBeAg以及HBV cccDNA的量并同时说明HBV在细胞内是完成完整复制表达的,采用染色质免疫共沉淀(ChIP)的方法检测与HBV cccDNA结合的组蛋白H3以及H3乙酰化水平的动态变化。结果显示,NIRF蛋白下调HBV标志物HBeAg、HBsAg的分泌以及HBV cccDNA的表达,表明其对HBV复制具有抑制作用;组蛋白H3及乙酰化的组蛋白H3都与HBV cccDNA的动态变化水平呈现相似的平行性,而NIRF蛋白也明显抑制组蛋白H3的表达水平和乙酰化水平。结论证实NIRF不仅能抑制HBV在肝癌细胞中的复制,而且能下调与HBV cccDNA结合的组蛋白H3和乙酰化组蛋白H3的表达。期待NIRF能为后续的HBV致病机理、HBV复制表观遗传学水平研究及有效抗病毒药物的研究与开发提供理论上的支持与帮助。
To study the replication of the hepatitis B virus (HBV) by NIRF (Np95/ICBP-90 like RING finger protein) and the histone H3 acetylation linked to the hepatitis B virus covalent loop-closed DNA (HBV cccDNA) The effect of the transfection was achieved by transfection of pGEM-HBV1.3+pFLAG, pGEM-HBV1.3+pFLAG-NIRF, and pGEM-HBV1.3 plasmids into HepG2 cells using lipofectamine. Western blot was used to detect the expression of NIRF protein. ELISA and RT-PCR were used to detect the amounts of HBsAg, HBeAg, and HBV cccDNA and also to show that HBV is completely expressed in cells. ChIP assay was used to detect histone H3 and H3 bound to HBV cccDNA. Dynamic changes in acetylation levels. The results showed that the down-regulation of HBV markers HBeAg and HBsAg and the expression of HBV cccDNA by NIRF protein showed that it had an inhibitory effect on HBV replication; both histone H3 and acetylated histone H3 were similar to the dynamic changes of HBV cccDNA Parallelism, while NIRF protein also significantly inhibits histone H3 expression and acetylation levels. Conclusions It is confirmed that NIRF not only inhibits the replication of HBV in hepatoma cells, but also down-regulates the expression of histone H3 and acetylated histone H3 bound to HBV cccDNA. It is expected that NIRF can provide theoretical support and assistance for the follow-up HBV pathogenesis, research on HBV replication epigenetics, and research and development of effective antiviral drugs.