扬州大学学报（农业与生命科学版）2023，Vol.44Issue(6)：62-69,8.DOI:10.16872/j.cnki.1671-4652.2023.06.008
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
摘要
Abstract
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 insert-ed 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 estab-lishment 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Key words
Getah virus/infectious clones/rescue of virus/reporter gene分类
农业科技引用本文复制引用
周景业,孟慧,鹿田原,牟春晓,陈振海..猪盖塔病毒感染性克隆构建及病毒拯救[J].扬州大学学报（农业与生命科学版）,2023,44(6):62-69,8.基金项目
江苏高校优势学科建设工程项目(PAPD) （PAPD）
江苏省高等学校大学生创新创业训练计划重点项目(202211117068Z) （202211117068Z）
