Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas - Universitat Politècnica de València (CSIC-UPV), 46011 Valencia, Spain.
STAR Protoc. 2022 Dec 16;3(4):101716. doi: 10.1016/j.xpro.2022.101716. Epub 2022 Sep 22.
Infectious clone technology is universally applied for biological characterization and engineering of viruses. This protocol describes procedures that implement synthetic biology advances for streamlined assembly of virus infectious clones. Here, I detail homology-based cloning using biological material, as well as SynViP assembly using type IIS restriction enzymes and chemically synthesized DNA fragments. The assembled virus clones are based on compact T-DNA binary vectors of the pLX series and are delivered to host plants by Agrobacterium-mediated inoculation. For complete details on the use and execution of this protocol, please refer to Pasin et al. (2017, 2018) and Pasin (2021).
传染性克隆技术广泛应用于病毒的生物学特性分析和工程改造。本方案描述了应用合成生物学方法实现病毒感染性克隆的简化构建的步骤。这里，我详细介绍了基于同源重组的克隆方法，以及利用 II 型限制性内切酶和化学合成 DNA 片段的 SynViP 组装方法。组装的病毒克隆基于紧凑的 pLX 系列 T-DNA 二元载体，并通过农杆菌介导的接种递送到宿主植物。如需了解本方案使用和实施的详细信息，请参考 Pasin 等人（2017 年，2018 年）和 Pasin（2021 年）的文献。
