貉源阿留申病毒感染性克隆的构建及病毒拯救  

Construction of infectious clone of raccoon dog and arctic fox amdoparvovirus and virus rescue

作　　者：彭倩文 赵永强 邵西群 王桂武[1] 张傲 陈立志[1] 章秀婷[1] PENG Qianwen;ZHAO Yongqiang;SHAO Xiqun;WANG Guiwu;ZHANG Ao;CHEN Lizhi;ZHANG Xiuting(Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130112,China;Northwest A&FUniversity,Yangling 712100,China;Jilin Agricultural Science and Technology University,Jilin 132101,China)

机构地区：[1]中国农业科学院特产研究所,吉林长春130112 [2]西北农林科技大学,陕西杨凌712100 [3]吉林农业科技学院,吉林吉林132101

出　　处：《中国兽医科学》2024年第7期853-858,共6页Chinese Veterinary Science

基　　金：吉林省自然科学基金项目(20190201169JC);中国农业科学院科技创新工程项目(CAAS-ASTIP-2021-ISAPS)。

摘　　要：旨在建立貉源阿留申病毒(RFAV)反向遗传系统,为在全基因水平上研究阿留申病毒的致病机制以及疫苗的研制提供平台。前期通过分段克隆成功获得RFAV中间编码区序列M1、M2、M3及末端序列信息。通过无缝克隆技术将RFAV中间编码区序列M1、M2、M3及人工合成的末端编码区序列构建到表达载体pBBSmaⅠ上,测序验证获得了全长质粒pBB-RFAV,将pBB-RFAV转染至F81细胞进行病毒拯救。通过细胞病变观察、PCR检测、qPCR检测病毒相对表达量、间接免疫荧光试验(IFA)、病毒粒子电镜观察共同验证拯救病毒。结果显示,细胞在转染后第48小时即可观察到明显的细胞病变,PCR检测到RFAV片段,qPCR鉴定发现拯救病毒可在F81细胞上稳定传代,增殖动态与水貂阿留申病毒(AMDV)相似,IFA结果显示感染细胞呈现特异荧光信号,通过电镜观察发现拯救病毒的粒子形态与AMDV已知形态特征基本一致。上述结果表明,构建了RFAV全基因组感染性克隆,并成功拯救出rRFAV,为深入研究阿留申病毒的病原学和免疫学奠定了基础。The aim was to establish a reverse genetic system for raccoon dog and arctic fox amdoparvovirus(RFAV)and to provide a platform for studying the pathogenic mechanisms of amdoparvoviruses and vaccine development at the whole gene level.Our laboratory has successfully obtained the sequence information of RFAV middle coding region(M1,M2,M3)and terminal sequences through segmental cloning.The sequences of the middle coding regions M1,M2,M3,and the synthetic terminal coding region sequences of RFAV were constructed into the expression vector p BBSmaⅠusing seamless cloning technology.The full-length plasmid p BB-RFAV was obtained,and this plasmid was then transfected into F81 cells for virus rescue.Verification of the rescued virus was conducted through observations of cytopathic effects,PCR detection,q PCR detection of the relative expression of the virus,indirect immunofluorescence assay(IFA)of r RFAV,and virion electron microscopy.Results indicated that signif cant cytopathic changes were observed in the cells at 48 hours,and RFAV fragments were detected by PCR.q PCR confirmed that the rescued virus could be stably passaged in F81 cells,exhibiting proliferation dynamics similar to that of Aleutian mink disease virus(AMDV).IFA results demonstrated specific fluorescent signals in infected cells,and the particle morphology of the rescued virus was consistent with the known morphological characteristics of AMDV according to electron microscopy.These findings highlight the successful construction of the whole-genome infectious clone of RFAV,leading to the successful rescue of the virus r RFAV.This achievement lays the foundation for further studies on the etiology and immunology of amdoparvoviruses.

关 键 词：貉源阿留申病毒 感染性克隆 病毒拯救 反向遗传操作系统 

分 类 号：S852.659.2[农业科学—基础兽医学]