PCV2/PCV3二价重组杆状病毒疫苗的构建与鉴定  

Construction and identification of PCV2/PCV3 bivalent recombinant baculovirus vaccine

作　　者：徐鹏[1,2] 姜宇航 许汪 宋利娜 李乐天 陈竞 郝鹏飞 金宁一 任林柱 李昌[2] XU Peng;JIANG Yuhang;XU Wang;SONG Lina;LI Letian;CHEN Jing;HAO Pengfei;JIN Ningyi;REN Linzhu;LI Chang(College of Animal Science,Jilin University,Changchun 130062,China;Research Unit of Key Technologies for Prevention and Control of Virus Zoonoses,Chinese Academy of Medical Sciences,Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130062,China)

机构地区：[1]吉林大学动物科学学院,吉林长春130062 [2]中国医学科学院人兽共患病毒病防控关键技术研究创新单元中国农业科学院长春兽医研究所,吉林长春130122

出　　处：《中国兽医学报》2021年第10期1915-1921,共7页Chinese Journal of Veterinary Science

基　　金：中国医学科学院医学与健康科技创新工程资助项目(2020-12M-5-001);国家自然科学基金面上资助项目(31772747);吉林省科技发展计划重点研发资助项目(20200402043NC)。

摘　　要：本试验旨在构建并获得共表达PCV2/PCV3 Cap蛋白的病毒样颗粒(virus-like particles,VLPs)。扩增PCV2 Cap及PCV3 Cap基因,将其克隆至pEASY-Blunt载体,分别命名为pEPC2、pEPC3,验证正确后将目的基因分别连接至杆状病毒双启动子表达载体pFastBaC-Dual,获得含有PCV2、PCV3 Cap基因的重组杆状病毒质粒pFBD-PC2-PC3。将该重组质粒转化至大肠杆菌DH10-BacTM感受态细胞,进行蓝白斑筛选与PCR鉴定,获得重组杆粒Bacmid-PC2-PC3。利用脂质体将Bacmid-PC2-PC3转染至SF9细胞进行病毒拯救,转染后72 h收取上清病毒液,并盲传3代。用其感染Sf9细胞,通过间接免疫荧光及Western blot验证目的蛋白是否表达;同时收集感染后的Sf9细胞上清,在电镜下观察是否形成VLPs。结果显示,成功构建了共表达PCV2/PCV3 Cap蛋白的重组质粒,并获得重组杆粒,拯救出重组杆状病毒。病毒感染Sf9细胞后,目的蛋白获得表达且具有反应原性,能够形成VLPs。本研究成功制备了共表达PCV2/PCV3 Cap蛋白的VLPs,为深入开展猪圆环病毒二价新型疫苗研发奠定了坚实基础。The purpose of this study is to obtain the Cap protein of porcine circovirus(PCV)with natural conformation and good reactogenicity.The PCV2 and PCV3 Cap genes were amplified and cloned into the pEASY-Blunt vector.Then,the target genes were cloned into the baculovirus expression vector pFastBaC-Dual(containing dual promoters),yielding the recombinant baculovirus plasmid pFBD-PC2-PC3 containing two Cap genes.The pFBD-PC2-PC3 was transformed into E.coli DH10-Bac^(TM) competent cells,followed by blue and white spot screening,resulting in recombinant Bacmid-PCV2-PCV3.Thereafter,the Bacmid-PCV2-PCV3 was transfected into Sf9 cells by liposome for virus rescue,generating rescued virus rBV-PC2-PC3.The expression of the target protein was verified by indirect immunofluorescence and Western blot.In addition,the virus particles were observed by the transmission electron microscope.The results showed that the recombinant plasmid coexpressing PCV2/PCV3 Cap protein was constructed successfully,and the recombinant baculovirus was rescued and the virus-like particles(VLP)could be observed under transmission electron microscopy.In this study,VLPs coexpressing PCV2/PCV3 Cap proteins were successfully prepared,which laid a foundation for the further development of new bivalent vaccine of porcine circovirus.

关 键 词：猪圆环病毒 重组杆状病毒 CAP蛋白 病毒样颗粒 

分 类 号：S855.3[农业科学—临床兽医学]