应用腺病毒载体系统将HBV/C全基因组转导入HepG2细胞的方法学研究

目的 运用腺病毒系统将HBV/C全基因组高效地导入肝癌细胞系,为研究HBV的复制及蛋白表达情况构建技术平台.方法 构建HBV/C基冈型毒株的1.3拷贝全基因序列,运用腺病毒系统(AdEasy),首先将1.3拷贝HBV全基因序列插入到穿梭载体pAdTrack,经测序证实插入序列的正确性后用Pme I酶切线性化重组穿梭质粒,转化Adeasier-I感受态细胞,在细菌细胞内发生同源重组,所得重组腺病毒质粒pAdEasy-HBV/C用PacI酶切线性化,用脂质体法转染293细胞来包装重组病毒.用适量感染复数的重组腺病毒感染HepG2细胞,并对感染细胞上清中的HBV DNA、HBeAg和HBsAg分别进行了定量检测.结果 成功运用腺病毒系统将1.3拷贝HBV全基因组高效率地导入HepG2细胞,导入的HBV能够运用自身的复制调控元件进行病毒的复制和蛋白表达.结论 运用AdEasy系统能够简便、快速、高效地将HBV全基因组导入肝癌细胞系,为抗病毒药物的筛选及评价提供了有效的技术平台.

Adenovirus-mediated transduction of HBV/C genome into HepG2 cell line

Objective To establish a method for efficient transfer of 1.3-fold HBV/C genome into HepG2 cell line using adenoviral vector system for studying the replication and antigen expression of HBV.Methods The 1.3-copy overlength genome of HBV genotype C was constructed and cloned into the shuttle vector pAdTrack.After confirmation of the constructed HBV genome by sequencing, the resultant plasmid linearized by digestion with Pme I was transformed into competent E.coli Adeasier-1 cells.The recombinants of pAdEasy-...