乙型肝炎病毒野生株和阿德福韦酯耐药变异株稳定表达细胞模型的建立和应用

Tetracycline-inducible replications of wild-type and an adefovir-dipivoxil-resistant hepatitis B virus in human liver cells

目的 构建稳定表达乙型肝炎病毒（HBV）野生株和rtE218G阿德福韦酯耐药变异株的人细胞模型，评价HBV野生株和rtE218G变异株的体外药物敏感性。方法 基于人肝细胞系HepG2，构建整合表达四环素调控元件（tTA）的工具细胞株；重组HBV野生株和rtE218G变异株HBV 1.2倍拷贝基因组至pTRE-Tight载体，与线性筛选标记物共转染tTA稳定表达细胞系，潮霉素筛选克隆，长期传代培养后，挑选受四环素严格调控表达的HBV高水平复制克隆，并对上述HBV野生株和变异株对阿德福韦酯的体外敏感性进行评价。结果 克隆筛选获得tTA稳定表达的细胞株HepG2-off23，构建了HBV野生株和rtE218G变异株四环素调控表达质粒pTRE-HBV-WT（野生型）和pTRE-HBV-E218G（变异型），分别稳定转染HepG2-off23细胞后，成功筛选得到基于HepG2的野生型和变异型HBV高水平可调控表达细胞株HepG2-tetHBV-WT（HBV野生株）和HepG2-tetHBV-E218G（rtE218G变异株）。培养144 h后，可检测到胞内HBV核心抗原（HBcAg）的高表达和病毒DNA复制中间体的高水平复制；且该细胞模型中的病毒复制可被培养体系中加入的四环素高效阻断，当四环素浓度为1 000 ng/ml时，Southern印迹检测不到复制中间体的存在。体外药物敏感性实验中，阿德福韦酯体外能够有效抑制HBV野生株的复制，半数抑制浓度（IC50）为（2.49±0.05）μmol/L，而rtE218G变异株表现出对阿德福韦酯的敏感性降低，其IC50为（6.49±0.09）μmol/L。结论 构建了基于四环素调控表达的HBV野生株和rtE218G变异株稳定复制细胞模型。HepG2-off23工具细胞系结合pTRE-HBV载体，可以较为方便地构建HBV不同变异株的稳定细胞模型，为HBV病毒学研究和抗病毒药物筛选模型的建立提供有力支持。

Objective To establish cell lines with inducible replications of wild-type or rtE218G, an adefovir-dipivoxil-resistant HBV mutant.Methods Tetracycline transactivator （tTA） was stably transfected into human liver cell line HepG2.1.2 folds of full-length of wild-type or rtE218G-mutated HBV genomes were cloned into the pTRE vector and cotransfected into the tTA-expressing cells with a linear selection marker for hygromycin, respectively. After hygromycin screening, clones with the highest levels of tetracycline-inducible HBV replications were selected. The obtained cell lines were further used to evaluate the in vitro sensitivity of rtE218G mutant to adefovir-dipivoxil.Results HepG2-off23, a HepG2-derived cell line with stable tTA expression was established. PTRE-based plasmids carrying wild-type HBV （pTRE-HBV-WT） or rtE218G mutant （pTRE-HBV-E218GHBV） were constructed. After stable transfection of the HBV constructs into HepG2-off23 cells, cell lines with robust and tetracycline-inducible replications of wild-type HBV （HepG2-tetHBV-WT） and rtE218G-mutated HBV （HepG2-tetHBV-E218G） were selected. In the two cell lines, high levels of viral core protein and DNA replication could be detected after 144 hours of culture, which could be potently inhibited when tetracycline was added into the medium. At the presence of 1 000 ng/ml of tetracycline, HBV replication intermediates were hardly detected by Southern blotting experiments. HBV mutant with rtE218G could independently confer resistance to adefovir in vitro. IC50 for HBV rtE218G mutant of adefovir was （6.49±0.09） μmol/L, which was significantly higher than that for wild type virus （2.49±0.05） μmol/L.Conclusion Wild-type and the rtE218G HBV mutant could be expressed and efficiently regulated by tetracycline in the established new cell lines. These cell lines could be useful tools for the HBV virology and anti-HBV drug screening studies.