Abstract：
The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.
摘要：
本研究旨在建立一种快速构建猪圆环病毒2型(PCV2)感染性克隆的方法。在这项研究中,我们通过无缝克隆技术构建了PCV2的圆形感染性克隆，使用临床分离的菌株PCV2-LX作为模板。同时,将该方法与常规限制性连接方法进行比较，重点研究了基因组的体外环化（自连接）过程和拯救病毒的生长特性。结果表明，该方法消除了分析和引入限制性内切酶位点的需要，从而避免了与传统的基于限制性内切酶的克隆步骤相关的复杂性。它提供了一个简单和快速的操作，能够更有效地编辑PCV2基因组。用这种方法构建的感染性克隆可以通过脂质体转染成功挽救,从而产生可以稳定传代的重组病毒。此外，通过这种方法拯救的重组病毒在PK-15细胞和3D4/31细胞(永生化猪肺泡巨噬细胞)中表现出增强的增殖能力.总之,本研究建立了一种新型的PCV2反向遗传系统，为PCV2基因工程疫苗的开发提供了新的策略。此外,它可作为构建其他新兴圆环病毒如PCV3和PCV4的感染性克隆的参考。
