猪盖塔病毒感染性克隆构建及病毒拯救  

Construction and rescue of an infectious clone of Getah virus

作　　者：周景业 孟慧 鹿田原 牟春晓 陈振海 ZHOU Jingye;MENG Hui;LU Tianyuan;MOU Chunxiao;CHEN Zhenhai(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2023年第6期62-69,共8页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：江苏高校优势学科建设工程项目(PAPD);江苏省高等学校大学生创新创业训练计划重点项目(202211117068Z)。

摘　　要：为研究盖塔病毒(GETV)致病机制,需搭建可靠的盖塔病毒(GETV)反向遗传操作平台。首先构建GETV/HuN1株的全长感染性克隆质粒,并在nsP4基因与C基因之间或E1基因与3′UTR之间插入亚基因组启动子26S和报告基因EGFP,将3个重组质粒分别转染至BHK-21细胞中,拯救病毒,鉴定重组病毒和外源基因稳定性,测定病毒滴度并绘制生长曲线。以重组质粒pACYC-177-GETV为模板进行RT-PCR扩增,结果显示,GETV每个蛋白基因均能正确扩增出相应条带。IFA鉴定结果显示,E2与6K蛋白抗体能与rGETV特异性结合。将EGFP基因分别插入到C基因的上游或E1基因的下游,产生报告病毒rGETV-EGFP/C和rGETV-EGFP/E1。在2种报告病毒感染的BHK-21细胞上均能观察到EGFP高表达,将报告基因插入E1基因下游,可保持其更高的稳定性。此外,生长曲线显示,重组病毒具有与亲代病毒相似的生长速度。综上,GETV反向遗传操作平台的成功建立为研究GETV蛋白功能和研发疫苗奠定了基础。To set up a reliable reverse genetic manipulation platform for Getah virus(GETV) to provide a valuable tool for investigating the pathogenesis of GETV. First, by constructing the full-length infectious clone plasmid of GETV/HuN1 strain, and inserting the subgenomic promoter 26S and the reporter gene EGFP between the nsP4 gene and C gene or between the E1 gene and 3' UTR, three recombinant plasmids were transfected respectively into BHK-21 cells to rescue the virus, identify the stability of recombinant virus and exogenous genes, determine the virus titers, and plot the growth curve. The results of RT-PCR amplification using the recombinant plasmid pACYC-177-GETV as a template showed that each protein gene of GETV could be correctly amplified. The results of immunofluorescence assay(IFA) showed that E2 and 6K protein antibodies could specifically bind to rGETV. The enhanced green fluorescent protein(EGFP) gene was inserted upstream of the Capsid gene or downstream of the E1 gene, generating the reporter viruses rGETV-EGFP/C and rGETV-EGFP/E1. High expression of EGFP could be observed in BHK-21 cells infected with both reporter viruses, and inserting the reporter gene downstream of the E1 gene maintained higher stability. In addition, the growth curve showed that the recombinant virus had a similar growth rate to the parental virus. These results demonstrate the successful establishment of the reverse genetic manipulation platform for GETV, laying the foundation for studying the protein function of GETV and developing vaccines.

关 键 词：盖塔病毒 感染性克隆 病毒拯救 报告基因 

分 类 号：S852.65[农业科学—基础兽医学]