摘要
本研究通过靶向设计2条sgRNA对AH2012/12全基因BAC重组质粒进行切割,将S-indel毒株的S基因质粒与AH2012/12基因BAC质粒转化至NEB 10-beta大肠杆菌,获得重组BAC质粒,转染至Vero细胞,拯救出S-indel毒株S基因替代AH2012/12毒株S基因的嵌合重组病毒。病毒基因序列分析、间接免疫荧光试验、空斑试验及生长曲线测定等证明该嵌合病毒株拯救成功,命名为rAH2012/12-S,本研究为PEDV新疫苗研发和快速应对新发PED防控奠定了重要基础。
In this study,two specifically sgRNAs were designed and utilized to cleave the full-gene BAC recombinant plasmid of AH2012/12.A recombinant BAC plasmid was obtained by transforming the S gene recombinant plasmid of the S-indel strain,and the AH2012/12 gene BAC plasmid into NEB 10-beta E.coli.Subsequently,transfection of the recombinant BAC plasmid into Vero cells resulted in the rescue of a chimeric AH2012/12 recombinant virus strain,wherein the S gene was replaced with that of the S-indel strain.Viral genome sequencing analysis,indirect immunofluorescence assays,plaque assays,and growth curve measurements collectively confirmed the successful rescue of this chimeric virus,designated as rAH2012/12-S.It lays an important foundation for the development of novel PEDV vaccines,and enables a more rapid response to the prevention and control of emerging PEDV variants in the future.
作者
尹丽萍
常长琳
范宝超
丁小燕
白娟
张海涛
李彬
姜平
YIN Liping;CHANG Zhanglin;FAN Baochao;DING Xiaoyan;BAI Juan;ZHANG Haitao;LI Bin;JIANG Ping(College of Veterinary Medicine,Nanjing A gricultural University,Nanjing 210095,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210095,China;Jiangsu Lihua A nimal Husbandry Co.,Ltd.,Changzhou 213100,China)
出处
《中国兽医科学》
北大核心
2025年第7期877-883,共7页
Chinese Veterinary Science
基金
国家生猪产业技术体系专项(CARS-35)
江苏省自主创新研发基金项目(CX(22)1011)。
关键词
猪流行性腹泻病毒
S基因
反向遗传
porcine epidemic diarrhea virus
S gene
reverse genetics
