NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China.
NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China; School of Public Health and Management, Weifang Medical University, Weifang, 261053, China.
J Biotechnol. 2020 Jan 10;307:193-201. doi: 10.1016/j.jbiotec.2019.11.009. Epub 2019 Nov 18.
Commonly used and well accepted approaches are lacking for site-directed modification of adenoviral vectors. Here, we attempt to introduce an easy-to-implement strategy for such purpose with an example of establishing a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR products of GFP expression cassette and plasmid backbone were fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA assembly. NdeI-digested fragment of pMDXE3GA was brought back to pKAd5 to form the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension K562 cells were investigated. Expression of target gene was significantly enhanced in both cell lines infected with HAdV5-XE3GA due to virus replication. However, propagation of virus could not sustain in culture of K562 cells. Shuttle plasmid pSh5RC-GFP was constructed to facilitate exchange of transgene. In summary, the strategy of combined DNA assembly and restriction-ligation cloning is functional, cost-effective and suitable for genetic modification of adenovirus.
目前缺乏用于腺病毒载体定点修饰的常用且被广泛认可的方法。在这里，我们尝试通过建立来自 pKAd5 质粒（人腺病毒 5 的感染性克隆）的复制型腺病毒载体系统的示例来实现这一目标，介绍一种易于实施的策略。GFP 表达盒和质粒骨架的 PCR 产物与 pKAd5 的 EcoRI/NdeI 消化片段融合，通过 DNA 组装生成修饰的中间质粒 pMDXE3GA。NdeI 消化的 pMDXE3GA 片段通过限制性连接克隆被带回 pKAd5，形成腺病毒质粒 pKAd5XE3GA。重组腺病毒 HAdV5-XE3GA 被拯救、扩增和纯化。在贴壁 HEp-2 和悬浮 K562 细胞中研究了 GFP 的表达和病毒的增殖。由于病毒复制，感染 HAdV5-XE3GA 的两种细胞系中靶基因的表达显著增强。然而，病毒在 K562 细胞培养中不能持续增殖。构建穿梭质粒 pSh5RC-GFP 以促进转基因的交换。总之，组合 DNA 组装和限制性连接克隆的策略是可行的、具有成本效益的，并且适用于腺病毒的遗传修饰。
