基于体外自连接的猪圆环病毒2型感染性克隆构建方法  

An in vitro self-ligation-based method for constructing infectious DNA clone of porcine circovirus type 2

作　　者：孙仁杰 杨永乐 王雅婷 谢荣辉[1] 张传亮[1] 方维焕[3] 李肖梁[3] 赵灵燕[1] SUN Renjie;YANG Yongle;WANG Yating;XIE Ronghui;ZHANG Chuanliang;FANG Weihuan;LI Xiaoliang;ZHAO Lingyan(Zhejiang Provincial Center for Animal Disease Control and Prevention,Hangzhou 311199,Zhejiang,China;Xianghu Laboratory,Hangzhou 311231,Zhejiang,China;Zhejiang Provincial Key Laboratory of Preventive Veterinary,College of Animal Sciences,Zhejiang University,Hangzhou 310058,Zhejiang,China)

机构地区：[1]浙江省动物疫病预防控制中心,浙江杭州311199 [2]湘湖实验室,浙江杭州311231 [3]浙江大学动物科学学院浙江省动物预防医学重点实验室,浙江杭州310058

出　　处：《生物工程学报》2024年第7期2333-2345,共13页Chinese Journal of Biotechnology

基　　金：浙江省自然科学基金(LQ23C180001);国家自然科学基金(32172819);浙江省动物预防医学重点实验室开放基金(ZPKLPVM2022KFKT001);浙江省“三农九方”科技协作项目(2022SNJF060,2023SNJF059)。

摘　　要：本研究旨在建立一种快速构建猪圆环病毒2型(porcine circovirus type 2,PCV2)感染性克隆的方法。以临床分离的PCV2 LX株为模板,利用DNA无缝克隆技术成功构建了PCV2环状感染性克隆。同时,在基因组体外自连接的构建过程以及拯救病毒的生长特性上,将本方法与常规酶切连接方法进行了比较。本方法无须分析和引入限制性内切酶位点,避免了传统酶切连接的繁琐,能更高效地进行PCV2基因组的编辑。构建的感染性克隆经脂质体转染细胞,成功拯救获得可以稳定传代的重组病毒,并且重组病毒在PK-15细胞和3D4/31细胞(猪肺泡巨噬细胞系)上具有更强的增殖能力。综上所述,本研究构建了一种基于体外自连接的PCV2反向遗传操作系统,为PCV2基因工程疫苗的研发提供新策略,为其他新发圆环病毒(PCV3和PCV4)感染性克隆的构建提供借鉴。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.

关 键 词：猪圆环病毒2型 感染性克隆 体外自连接 病毒拯救 

分 类 号：S852.651[农业科学—基础兽医学]