旨在通过缺失牛结节性皮肤病病毒(LSDV)ORF123基因,分析其增殖能力,为LSDV ORF123功能研究奠定基础.本研究利用同源重组,以ORF123基因作为靶标,增强型绿色荧光蛋白(EGFP)为筛选标记,采用融合PCR方法,扩增同源左右臂序列以及EGFP基因表达框,克隆至pUC-19T载体,构建基因缺失转移载体pH5-LSDV△ORF12 3-EGFP 质粒.然后将 pH5-LSDV△ORF123-EGFP 质粒转染至 Vero 细胞,并感染 LSDV/CHA/FJ/2021毒株,利用蚀斑、梯度稀释和挑取绿色荧光单克隆细胞筛选纯化重组毒株,并鉴定其遗传稳定性和增殖能力.结果显示,利用EGFP筛选标记,通过多次挑斑筛选纯化获得ORF123基因缺失的重组病毒株LSDV△ORF123-EGFP.该重组病毒至少在8代细胞传代中能稳定表达绿色荧光蛋白,接种MDBK细胞后绘制一步增殖曲线表明该重组病毒滴度略低于亲本毒株.本研究成功获得ORF123基因缺失的重组病毒株LSDV△ORF123-EGFP,为LSDV ORF123蛋白生物学功能研究以及减毒活疫苗的研制奠定基础.
Construction and Growth Characteristics of ORF 123 Deleted Lumpy Skin Disease Virus Strain
The aim of this study was to analyze the growth characteristics of the Lumpy skin dis-ease virus(LSDV)strain with the ORF123 gene deleted,which laid a foundation for the func-tional research of LSDV ORF123.ORF123 was used as the target gene and enhanced green fluo-rescent protein(EGFP)was used as the screening marker,the homologous left and right arm se-quences and the EGFP gene expression frame were amplified and fused by overlapping PCR and cloned into pUC-19T vector to constructpH5-LSDV△ORF123-EGFP.The plasmid pH5-LSDV△ORF123-EGFP was then transfected into Vero cells and after that the cells were infected with the LSDV/CHA/FJ/2021 strain.The recombinant virus strain LSDV△ORF123-EGFP was purified through multiple plaque screening using EGFP as a screening markers,and its viral ge-netic stability and growth characteristics were characterized.The results revealed that the recom-binant virus was able to stably express green fluorescent protein across at least 8 cell passages and the one-step growth curve following MDBK cell inoculation indicated that the titer of the recom-binant virus was slightly lower than that of the parental strain.In conclusion,the recombinant virus strain LSDV△ORF123-EGFP was successfully obtained,which laid a foundation for the study of the biological function of LSDV ORF123 protein and the development of a live attenuated LSDV vaccine.
万方数据
维普
