乙型肝炎病毒表达载体pHY106在筛选抗病毒药物中的意义

目的应用新的HBV表达载体,建立体外筛选临床抗HBV药物的方法。方法克隆对拉米夫定耐药的CHB患者体内HBV全基因组,然后将其亚克隆到HBV真核表达载体pHY106,体外转染Huh7细胞,检测转染后不同时间HBsAg、HBeAg、HBV DNA及HBV复制中间体水平。分析拉米夫定和阿德福韦对HBV基因表达和复制的抑制作用,指导临床用药。结果成功构建全部8个HBV临床分离株全基因组真核表达质粒,HBV聚合酶基因YMDD有5个产生YVDD突变,3个产生YIDD突变。该质粒上HBV基因能在Huh7细胞中进行复制和表达,拉米夫定不能体外抑制HBV复制,阿德福韦抑制了HBV在Huh7细胞中的复制,抑制程度与药物浓度相关。阿德福韦在患者体内也抑制了HBV的复制。结论新建立的体外筛选抗HBV药物的方法能用于临床快速筛选抗HBV药物,对CHB的治疗用药具有指导意义。

Significance of novel HBV expression vectors in selecting antiviral drugs in clinical therapy

OBJECTIVE: To establish a new method for rapidly selecting anti-hepatitis B virus drugs in clinical therapy. METHODS: The full-length hepatitis B virus (HBV) genomes from 8 patients with chronic hepatitis B (CHB) were generated by polymerase chain reaction (PCR). All patients were resistant to lamivudine therapy. Their HBV DNA fragments were inserted into Sap I site of pHY106 eukaryotic expression vector separately. The recombinant plasmids containing 1.1 copies of HBV genome were transfected into Huh7 cell line; the levels of HBsAg, HBeAg and HBV DNA in supernatants of Huh7 cells were measured by ELISA and real-time quantitative PCR, and intracellular HBV replicative intermediates were detected by Southern blot. Antiviral effects of lamivudine and adefovir were evaluated in this vitro system. RESULTS: The 8 recombinant plasmids containing a full-length genome of clinical HBV isolates could replicate and be expressed in Huh 7 cells. There were 6 isolates with polymerase YVDD mutations and 2 isolates with polymerase YIDD mutations. Adefovir, but not lamivudine, inhibited the HBV replication and gene expression in vitro. Furthermore, adefovir inhibited HBV replication in these CHB patients. CONCLUSION: The method described here enables a rapid selection of anti-HBV drugs in clinical therapy and is very useful in antiviral therapy for CHB patients.