【摘要】 基于转化相关重组（transformation-associated recombination, TAR）技术，通过将绿色荧光蛋白（green fluorescent protein, GFP）标记基因插入到猪传染性胃肠炎病毒（transmissible gastroenteritis virus, TGEV）的ORF3基因位点，成功构建了TGEV黑龙江流行株的感染性cDNA克隆。将感染性克隆转染到猪睾丸细胞（swine testis cells, ST）后，可以高效拯救重组病毒rTGEV-GFP。体外生长特性、复制动力学等试验确定拯救出病毒的生物学特性和复制等特性与亲本毒株无显著差异（P>0.05）。此外，借助拯救重组病毒表达的GFP报告基因，确定了瑞德西韦抗TGEV效力，其IC₅₀可达（1.230±0.010）μmol/L。综上，本试验利用TAR技术构建了TGEV基因组感染性克隆，显示出该技术可高效和准确地用于大基因组RNA病毒的cDNA片段组装，并可以很好地用于替代体外连接、靶向RNA重组技术、细菌人工染色体（bacterial artificial chromosome, BAC）等传统技术；获得表达GFP报告基因的重组病毒将为深入探讨TGEV致病机制、研制药物、进行中和抗体评价及新型疫苗开发提供可视化平台。更多还原

【Abstract】 In this study,we employed transformation-associated recombination（TAR） technology to insert the green fluorescent protein（GFP） reporter gene into the ORF3 gene locus of transmissible gastroenteritis virus（TGEV） and successfully generate an infectious DNA clone of the TGEV Heilongjiang epidemic strain.Transfection of swine testicular（ST） cells with this clone rescued the recombinant virus rTGEV-GFP efficiently.Through experiments such as in vitro growth characteristics and replication kinetics,it has been determined that the rescued virus exhibits no significant differences in biological characteristics and replication ability compared to the parental strain（P>0.05）.Besides,utilizing the GFP reporter,we further demonstrated the antiviral efficacy of remdesivir against TGEV,with an IC₅₀ value of（1.230±0.010） μmol/L.To our knowledge,this study represents the first application of TAR technology to construct an infectious clone of the TGEV genome,highlighting its superiority over conventional methods such as in vitro ligation,targeted RNA recombination,or bacterial artificial chromosome（BAC） systems for assembling large RNA virus genomes.The GFP-tagged recombinant virus provides a versatile platform for elucidating TGEV pathogenesis,screening antiviral compounds,evaluating neutralizing antibodies,and developing next-generation vaccines.更多还原