牛结节性皮肤病病毒ORF123基因缺失重组病毒的构建及增殖能力分析  

Construction and Growth Characteristics of ORF123 Deleted Lumpy Skin Disease Virus Strain

作　　者：王芳萍 任善会 郜晓虹 杨雪[1] 李积雲 王相伟 殷相平[2] 孙跃峰[2] 陈豪泰[2] 万学瑞[1] WANG Fangping;REN Shanhui;GAO Xiaohong;YANG Xue;LI Jiyun;WANG Xiangwei;YIN Xiangping;SUN Yuefeng;CHEN Haotai;WAN Xuerui(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Qinghai Academy of Science and Technology,Xining 810003,China)

机构地区：[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

出　　处：《畜牧兽医学报》2024年第10期4530-4541,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(32302850);甘肃省科技计划资助(22JR5RA035);甘肃省科技重大专项(22ZD6NA001);中国农业科学院兰州兽医研究所基本科研业务费(1610312021008)。

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-LSDVΔORF123-EGFP质粒。然后将pH5-LSDVΔORF123-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LSDVΔORF123-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LSDVΔORF123-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。The aim of this study was to analyze the growth characteristics of the Lumpy skin disease virus(LSDV)strain with the ORF123 gene deleted,which laid a foundation for the functional research of LSDV ORF123.ORF123 was used as the target gene and enhanced green fluorescent protein(EGFP)was used as the screening marker,the homologous left and right arm sequences and the EGFP gene expression frame were amplified and fused by overlapping PCR and cloned into pUC-19T vector to construct pH5-LSDVΔORF123-EGFP.The plasmid pH5-LSDVΔORF123-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 LSDVΔORF123-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 LSDVΔORF123-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.

关 键 词：牛结节性皮肤病病毒 ORF123基因 增殖能力 同源重组 重组病毒 

分 类 号：S852.654[农业科学—基础兽医学]