南京农业大学学报2025，Vol.48Issue(6)：1335-1342,8.DOI:10.7685/jnau.202407030
表达绿色荧光蛋白的A型人源呼吸道合胞病毒反向遗传系统的构建
Construction of reverse genetic system of human respiratory syncytial virus type A expressing green fluorescent protein
摘要
Abstract
[Objectives]In order to better study the pathogenesis of Human respiratory syncytial virus(HRSV)and facilitate its application,a reverse genetic operating system of HRSV-A2 strain was constructed in this study.Green fluorescent protein(GFP)was inserted into the HRSV-A2 genome to construct recombinant HRSV-A2 expressing GFP.[Methods]The full-length plasmid of HRSV-A2 strain was constructed using bacterial artificial chromosome(BAC)as the carrier skeleton,and GFP was inserted between P and M genes on this basis.Four helper plasmids expressing HRSV N,P,M2-1 and L protein were constructed using pCI as vectors,and the five plasmids were co-transfected into BHK21-T7 cell line expressing T7 RNA polymerase for virus rescue.[Results]rBAC-RSV-A2 recombinant virus and RBAC-RSV-A2-P-GFP-M recombinant reporter virus RNA were extracted,and whole genome sequencing was performed by RT-PCR.The results showed that the recombinant virus was successfully constructed.In the continuous passage of F1-F9,the green fluorescence was expressed,and the fluorescence disappeared in the F10 generation without cytopathy.The growth curve of the recombinant virus showed that the growth trend of the two recombinant viruses was roughly the same as that of the original virus,indicating that the insertion of foreign genes did not change the growth characteristics of the virus itself.The plaque observation and staining results showed that the plaque of the recombinant virus was smaller than that of the parent virus,indicating a decrease in virulence of the recombinant virus.[Conclusions]In this study,we successfully constructed an infectious clone of HRSV-A2 with BAC vector as the skeleton,and obtained recombinant HRSV virus that could express green GFP.The efficient and stable HRSV reverse genetics system established by the institute provided a useful technical platform for the research of HRSV pathogenic mechanism,vaccine and antibody drug development in China.关键词
人呼吸道合胞病毒/绿色荧光蛋白/反向遗传系统/重组病毒Key words
human respiratory syncytial virus/green fluorescent protein/reverse genetic system/recombinant virus分类
农业科技引用本文复制引用
倪思睿,简书令,余飞,易若男,武小琰,鲍恩东..表达绿色荧光蛋白的A型人源呼吸道合胞病毒反向遗传系统的构建[J].南京农业大学学报,2025,48(6):1335-1342,8.基金项目
江苏省农业科技自主创新资金项目[CX(22)2016] （22）
南京农业大学校企合作项目(技术服务类) （技术服务类）
