Construction and Characterization of a Hepatitis B Virus Replicon

建立复制细胞肝炎 B 病毒(HBV ) 当模特儿并且在抗病毒的药评估决定它的应用程序，我们构造了表情包含了 HBV 染色体的 1.3 个拷贝，并且在 Huh7 房间在短暂 transfection 以后测量了病毒的复制的水平的 plasmid。我们然后观察了抗病毒的药管理的效果。HBV (ayw ) 基因碎片的 1.3 褶层被 PCR 和限制 endonuclease 消化克隆进 pCR2.1。recombinant plasmid 是进 Huh7 房间， HBsAg， HBeAg 和 HBV 的短暂 transfected 在 Huh7 房间的上层清液的 DNA 被 ELISA 和即时 PCR 分别地测量；细胞内部的 HBV replicative 中介和细胞内部的 HBV 抄本被南部的污点和北污点分别地检测。adefovir 的抗病毒的效果，新奇 anti-HBV 核苷酸类似物，在这个细胞的模型系统被评估。结果显示 HBV replicon 的 recombinant plasmid 成功地被构造；在 plasmid pHBV1.3 带的 HBV 染色体能高效地复制并且被表示在哈 7 个房间， adefovir 能在这个细胞的模型，和抑制禁止 HBV 复制是剂量依赖者。结论是 HBV replicon，能在 hepatoma 房间高效地开始病毒的复制，可以是在 HBV 复制和抗病毒的药的学习的一个有用工具。关键词肝炎 B 病毒 - 传染 replicon - 表示向量 CLC 数字 R373 基础条款：国家自然科学基础(No.30271170， No.30170889 ) 。

To establish a replication cellular model of hepatitis B virus （HBV） and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV（ayw） gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was trans ient transfected into Huh7 cells, HBsAg, HBeAg and HBV DNA in supematant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.