乙型肝炎病毒表达载体pHY106在筛选抗病毒药物中的意义

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

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、HBVDNA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。

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.