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.

中文翻译：

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通过结合DNA组装和限制性连接克隆对腺病毒载体进行定点修饰。

腺病毒载体的定点修饰缺乏常用和公认的方法。在这里，我们尝试引入一个易于实施的策略，以从pKAd5质粒（人腺病毒5（HAdV-5）的感染性克隆）建立具有复制能力的腺病毒载体系统为例。将GFP表达盒和质粒主链的PCR产物与EcoRI / NdeI消化的pKAd5片段融合，以通过DNA组装产生修饰的中间质粒pMDXE3GA。通过限制性连接克隆将NdeI消化的pMDXE3GA片段带回到pKAd5，以形成腺病毒质粒pKAd5XE3GA。拯救，扩增和纯化重组腺病毒HAdV5-XE3GA。研究了GFP在粘附的HEp-2和悬浮K562细胞中的表达以及病毒的传播。由于病毒复制，在感染HAdV5-XE3GA的两种细胞系中靶基因的表达均显着增强。但是，病毒的繁殖不能在K562细胞的培养中持续。构建穿梭质粒pSh5RC-GFP以促进转基因的交换。总之，结合DNA组装和限制性-连接克隆的策略是功能性的，成本有效的并且适合于腺病毒的遗传修饰。