一种基于HDR-CRISPR/Cas9技术快速构建重组鸭肠炎病毒的方法的建立  

Development of a Method for Rapid Construction of Recombinant Duck Enteritis Virus Based on HDR-CRISPR/Cas9 Technology

作　　者：贾文凤 蒋香香 陶慧丽 王安平[1,2] 吴植[1,2] 朱善元[1,2] JIA Wenfeng;JIANG Xiangxiang;TAO Huili;WANG Anping;WU Zhi;ZHU Shanyuan(Engineering Technology Research Center for Modern Animal Science and Novel Veterinary Pharmaceutic Development,Jiangsu Key Laboratory of Veterinary Bio-pharmaceutical High Technology Research,Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China)

机构地区：[1]江苏农牧科技职业学院,江苏省兽用生物制药高技术研究重点实验室,江苏现代畜牧与新兽药工程技术中心,泰州225300 [2]江苏农牧科技职业学院,泰州225300

出　　处：《中国畜牧兽医》2025年第1期298-309,共12页China Animal Husbandry & Veterinary Medicine

基　　金：江苏省高等学校自然科学研究面上项目(24KJB230003);江苏农牧科技职业学院校级科研资助项目(NSF2023CB04);江苏农牧科技职业学院科技创新团队资助项目(NSF2023TC02);2023年江苏省职业院校学生创新创业培育计划项目(G-2023-0728)。

摘　　要：[目的]本研究通过优化试验条件,建立基于HDR-CRISPR/Cas9基因编辑技术快速、准确地将外源基因定向插入鸭肠炎病毒(Duck enteritis virus, DEV)基因组的方法,为以DEV为载体的重组疫苗的研制奠定基础。[方法]分离并扩繁DEV疫苗株,测定其病毒滴度。根据DEV疫苗株UL26、UL27基因间非编码区序列,设计合成向导RNA(gRNA),经双链退火获得目的片段,通过基因克隆技术将其插入PX459-V2.0载体获得gRNA-Cas9质粒。同时,通过常规基因克隆技术将增强型绿色荧光蛋白(EGFP)报告基因插入PVAX-1载体,构建含有真核表达盒P_(CMV)-EGFP-BGH pA的重组表达质粒。以DEV疫苗株基因组为模板,扩增gRNA上、下游同源臂序列,通过融合PCR将同源臂序列与真核表达盒P_(CMV)-EGFP-BGH pA进行连接并克隆至PUC19载体获得供体质粒PUC19-UP-EGFP-DOWN。采用先转染质粒后感染亲本DEV的策略构建重组病毒rDEV-EGFP,通过控制单一变量法优化重组病毒构建的试验条件,根据不同条件下绿色荧光蚀斑数量确定最佳条件。利用有限稀释法筛选并纯化表达绿色荧光的蚀斑,获得重组病毒rDEV-EGFP,并对其进行遗传稳定性及体外复制能力的评估。[结果]PCR扩增及测序结果显示,成功构建靶向DEV基因组UL27与UL26基因间非编码区序列的gRNA-Cas9质粒及包含EGFP真核表达盒的供体质粒。鸡胚成纤维细胞(chick embryo fibroblast, CEF)中转染gRNA-Cas9及供体质粒后感染DEV,在荧光显微镜下可观察到绿色荧光蚀斑,表明成功利用HDR-CRISPR/Cas9基因编辑技术将EGFP报告基因敲入DEV基因组。优化后的最佳试验条件为:DEV感染复数(MOI)为0.2、gRNA-Cas9质粒与供体DNA转染比例为1∶2、供体DNA的转染形式为DNA片段、转染与感染时间间隔为6 h、重组病毒收取时间为DEV感染后48 h,优化后EGFP报告基因的敲入效率显著提高(P<0.05)。重组病毒rDEV-EGFP在CEF中连续传代15次,EGFP真核表达盒仍稳定整合�[Objective]This study was aimed to establish a rapid and accurate method for targeted insertion of exogenous genes into the Duck enteritis virus(DEV)genome using the HDR-CRISPR/Cas9 gene editing technology under optimized conditions,so as to lay the foundation for the development of recombinant vaccines utilizing DEV as a vector.[Method]The DEV vaccine strain was isolated,propagated,and the viral titer was determined.The gRNA was designed and synthesized based on the non-coding sequence between UL 26 and UL 27 genes,then inserted into PX459-V2.0 to form the gRNA-Cas9 plasmid.Simultaneously,the enhanced green fluorescent protein(EGFP)reporter gene was inserted into the PVAX-1 vector via conventional gene cloning techniques to construct a recombinant expression plasmid containing the eukaryotic expression cassette P CMV-EGFP-BGH pA.Using the DEV genome as a template,upstream and downstream homologous arm sequences of the gRNA target site were amplified,fused with P CMV-EGFP-BGH pA,and cloned into PUC19 to obtain donor plasmid PUC19-UP-EGFP-DOWN.Recombinant virus rDEV-EGFP was constructed by transfecting plasmid and infecting parent DEV.The experimental conditions for the construction of the recombinant virus were optimized by controlling single variable method,and the optimal conditions were determined based on the number of green fluorescent plaques formed under different conditions.The plaques expressing green fluorescence were screened and purified using the limited dilution method to obtain the recombinant virus rDEV-EGFP.Subsequently,the genetic stability and in vitro replication ability of the recombinant virus rDEV-EGFP were evaluated.[Result]PCR amplification and sequencing results showed that the gRNA-Cas9 plasmid targeting the non-coding region between UL 27 and UL 26 genes of DEV genome and the donor plasmid containing EGFP eukaryotic expression box were successfully constructed.The chick embryo fibroblast(CEF)was infected with DEV after being transfected with gRNA-Cas9 and donor plasmid,and green fluore

关 键 词：鸭肠炎病毒 CRISPR/Cas9基因编辑 重组载体 

分 类 号：S852.657[农业科学—基础兽医学]