携带乙肝核心抗原基因复制缺陷型重组腺病毒的高效制备和表达

Construction of replication-deficient recombinant adenoviral vector carrying HBcAg gene and its expression in vitro

目的构建表达乙肝病毒核心抗原(HBcAg)的复制缺陷型重组腺病毒载体。方法扩增乙肝病毒(HBV)C基因片段,亚克隆到穿梭质粒pAdTrack-CMV上,与5型腺病毒骨架质粒pAdeasy-1共同电转化到大肠杆菌BJ5183内进行同源重组,经卡那霉素抗性筛选和酶切鉴定筛选出携带HBVC区基因的重组腺病毒载体,用脂质体包裹PacⅠ酶切线性化的重组质粒,转染到293细胞内进行重组腺病毒的包装。体外转染真核细胞,通过示踪基因绿色荧光蛋白表达的观察、RT-PCR和ELISA检测目的基因的表达。结果成功获得重组腺病毒质粒pAd-HBc。重组质粒pAd-HBc导入293细胞,经过包装和二次扩大培养获得了具有感染能力的重组腺病毒颗粒Ad-HBc,其病毒滴度达5×109pfu/mL,并能在真核细胞中有效表达目的基因。结论成功构建表达HBcAg复制缺陷型重组腺病毒载体,为进一步开展HBV基因治疗研究提供实验基础。

[Objective] To construct recombinant adenoviral vector carrying HBcAg gene by homologous recombination in bacteria and to detect its expression in vitro. [Methods] HBV C genes were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV. Then the resultant pAdTrack-CMV-HBc was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBV C gene （pAd-HBc） was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells. Several kinds of mammal cells （293 cells, Vero cells, HepG2 and MSCs） were infected with adenoviruses and the expression of HBcAg was detected by RT-PCR and FACS in vitro. [Results] The adenoviral plasmids pAd-HBc were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease Pac I analyses. The recombinant adenoviruses Ad-HBc were packaged successfully in 293 cells. The titer of Ad-HBc was up to 5x109 pfu/mL after the second generation of proliferation in 293 cells. HBcAg were expressed efficiently in mammal cells after infection. [Conclusion] The recombinant adenoviruses expressing HBcAg were constructed successfully and can be used in further study of gene therapy for HBV.