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

doi:10.16431/j.cnki.1671-7236.2025.01.027

摘要/Abstract

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在UL26与UL27基因之间的非编码区,具有良好的遗传稳定性。生长曲线结果显示,重组病毒rDEV-EGFP在CEF中的复制能力与DEV疫苗株无明显差异,生长趋势一致,具有良好的体外复制能力。【结论】本研究建立了一种基于HDR-CRISPR/Cas9基因编辑技术快速构建重组DEV的方法,成功构建了具有良好遗传稳定性及体外复制能力的重组病毒rDEV-EGFP,为重组DEV载体疫苗候选株的研制提供了理论依据及技术平台。

关键词: 鸭肠炎病毒; CRISPR/Cas9基因编辑; 重组载体

Abstract: 【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 UL26 and UL27 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 UL27 and UL26 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 fluorescence plaque could be observed under fluorescence microscope.The results showed that the EGFP reporter gene was successfully inserted into DEV genome using HDR-CRISPR/Cas9 gene editing technology.The optimal experimental conditions were as follows:Multiplicity of infection (MOI) of DEV was 0.2,the transfection ratio of gRNA-Cas9 plasmid to donor DNA was 1∶2,the transfection form of donor DNA was DNA fragment,the interval between transfection and infection was 6 h,and the collection time of recombinant virus was 48 h after DEV infection.After optimization,the knockin efficiency of EGFP reporter gene was significantly increased (P ＜ 0.05).Recombinant virus rDEV-EGFP was passed in CEF for 15 consecutive passages,and the EGFP eukaryotic expression box was still stably integrated in the non-coding region between UL26 and UL27 genes,showing good genetic stability.The results of growth curve showed that the replication ability of recombinant virus rDEV-EGFP in CEF was not significantly different from that of the DEV vaccine strain,and the growth trend was consistent,indicating good in vitro replication ability. 【Conclusion】 In this study,a method for rapidly constructing recombinant DEV based on HDR-CRISPR/Cas9 gene editing technology was established.Recombinant virus rDEV-EGFP with good genetic stability and in vitro replication ability was successfully constructed,which provided a theoretical basis and technical platform for the development of recombinant DEV vector vaccine candidate strains.

Key words: Duck enteritis virus; CRISPR/Cas9 gene editing; recombinant vector

中图分类号:

S852.65⁺7

贾文凤, 蒋香香, 陶慧丽, 王安平, 吴植, 朱善元. 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[J]. 中国畜牧兽医, 2025, 52(1): 298-309.

JIA Wenfeng, JIANG Xiangxiang, TAO Huili, WANG Anping, WU Zhi, ZHU Shanyuan. Development of a Method for Rapid Construction of Recombinant Duck Enteritis Virus Based on HDR-CRISPR/Cas9 Technology[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 298-309.

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