Construction and Growth Characteristics of ORF 123 Deleted Lumpy Skin Disease Virus Strain
王芳萍 1任善会 2郜晓虹 2杨雪 1李积雲 3王相伟 2殷相平 2孙跃峰 2陈豪泰 2万学瑞1
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作者信息
1. 甘肃农业大学动物医学院,兰州 730070
2. 中国农业科学院兰州兽医研究所,兰州 730046
3. 青海省科学技术信息研究所,西宁 810003
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摘要
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.
Abstract
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.
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