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 Laboratory Medicine, Weifang Medical University, Weifang 261053, China.
Viruses. 2022 Mar 6;14(3):546. doi: 10.3390/v14030546.
Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.
基因治疗和疫苗开发需要更多新型腺病毒载体。在这里，我们尝试为该领域经验较少的研究人员提供基于限制酶切和 Gibson 组装构建腺病毒载体的策略。限制酶切和 Gibson 组装是一种将限制酶切和 Gibson 组装相结合的方法，通过该方法获得的质粒的大部分来自于消化后的 DNA 片段，而不是 PCR 产物。在本研究中，我们展示了限制酶切和 Gibson 组装在操作猿猴腺病毒 1 (SAdV-1) 基因组方面的能力。通过 Gibson 组装，将质粒骨架的 PCR 产物与 SAdV-1 基因组 DNA 结合，构建了一个感染性克隆质粒 pKSAV1。在中间质粒分离、修饰和恢复的步骤中重复进行限制酶切和 Gibson 组装。用限制性内切酶消化线性化产生的腺病毒质粒，并转染包装 293 细胞以拯救 E3 缺失的复制型有效 SAdV1XE3-CGA 病毒。有趣的是，SAdV1XE3-CGA 可以在人慢性髓性白血病 K562 细胞中增殖。同样对 E1 区域进行修饰，生成 E1/E3 缺失的复制缺陷型病毒 SAdV1-EG。SAdV1-EG 对贴壁哺乳动物细胞具有适度的基因转移能力，与人类腺病毒 5 对照载体相比，它能有效地转导悬浮细胞。限制酶切和 Gibson 组装易于使用，无需特殊的实验材料和仪器即可操作。它在每一步都具有高效性和可验证的结果。更重要的是，限制酶切和 Gibson 组装使建立的载体系统具有可修饰性、可升级性和可持续发展性，它可以作为腺病毒合成生物学的指导方法或策略。
