猪瘟病毒E2基因部分表位区重组猪伪狂犬病病毒的构建及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2025.04.033

摘要/Abstract

摘要： 【目的】猪瘟病毒(Classical swine fever virus,CSFV)和猪伪狂犬病病毒(Pseudorabies virus,PRV)是当前影响生猪产业发展的两种重要病原，目前无高效的药物治疗方法，主要依靠疫苗接种进行预防。本试验旨在构建含有CSFV E2基因部分表位区的重组PRV毒株，并探究其生物学特性，为开发同时预防CSFV和PRV的二联疫苗提供参考。【方法】首先使用基因工程技术将包含CSFV E2蛋白B/C/D/A 4个表位区的一段基因插入pG-EGFP重组质粒的BamHⅠ位点，构建重组质粒pG-E2AD-EGFP；然后将质粒转染已接种PRV三基因缺失毒株rPRV NY-gE^-/gI^-/TK^-的ST细胞，在ST细胞内发生同源重组，利用蚀斑纯化法拯救出带绿色荧光蛋白的重组病毒rPRV-E2AD-EGFP。使用CRISPR/Cas9敲除载体敲除EGFP基因，经蚀斑纯化得到无荧光的重组病毒rPRV-E2AD。利用PCR扩增和Western blotting检测重组病毒rPRV-E2AD的E2基因A-D表位区表达情况。通过半数组织培养感染剂量(TCID₅₀)法检测不同理化性质处理后病毒增殖情况，对重组病毒培养特性、理化特性进行评价。【结果】通过细胞内同源重组和病毒蚀斑纯化，得到了同时表达CSFV E2AD抗原和EGFP的重组病毒rPRV-E2AD-EGFP。通过CRISPR/Cas9技术敲除EGFP基因，得到了无EGFP基因表达的重组病毒rPRV-E2AD。Western blotting检测结果显示，该毒株表达蛋白大小约27 ku，与预期CSFV E2AD抗原大小一致。rPRV-E2AD毒株与亲本株在ST、IPEC-J2、PK-15、Vero细胞中生长特性基本一致，均在感染后12 h引起细胞融合，36 h出现细胞脱落。理化特性检测结果显示，重组毒株与亲本株在相同理化条件处理下，培养特性相似。【结论】本研究成功获得重组病毒rPRV-E2AD，且外源基因不影响亲本株的增殖特性，试验结果为研发重组CSFV E2基因活载体疫苗提供了候选毒株，也为PRV活载体疫苗的开发提供了研究基础。

关键词: 猪瘟病毒(CSFV); 伪狂犬病病毒(PRV); 重组病毒; E2基因

Abstract: 【Objective】 Classical swine fever virus (CSFV) and porcine Pseudorabies virus (PRV) are two important pathogens affecting the development of current swine industry.Currently,there is no effective drug treatment,and the prevention mainly depends on vaccination.The aim of this study was to construct a recombinant PRV strain containing part of the epitope region of CSFV E2 gene and explore its biological characteristics,so as to provide reference for the development of dual vaccine against CSFV and PRV simultaneously.【Method】 Firstly,a gene segment containing the 4 epitope regions of B/C/D/A of CSFV E2 protein was inserted into the BamHⅠ site of the pG-EGFP recombinant plasmid,and the recombinant plasmid pG-E2AD-EGFP was constructed.The plasmid was then transfected into ST cells inoculated with PRV tri-gene deletion strain rPRV NY-gE^―/gI^―/TK^― and homologous recombination occurred in ST cells.The recombinant virus rPRV-E2AD-EGFP with green fluorescent protein was rescued by the plaque purification.The EGFP gene was knocked out using CRISPR/Cas9 knockout vector,and the non-fluorescent recombinant virus rPRV-E2AD was purified by plaque.The expression of A-D epitopes of E2 of recombinant virus rPRV-E2AD was detected by PCR amplification and Western blotting.The proliferation of the virus after treatment with different physicochemical properties was detected by 50% tissue culture infective dose (TCID₅₀) method,and the culture and physicochemical properties of the recombinant virus were evaluated.【Result】 Recombinant virus rPRV-E2AD-EGFP expressing both CSFV E2AD antigen and EGFP was obtained through intracellular homologous recombination and viral plaque purification.EGFP gene was knocked out by CRISPR/Cas9 technique,and recombinant virus rPRV-E2AD without EGFP gene expression was obtained.Western blotting results showed that the protein size of this strain was about 27 ku,which was consistent with the CSFV E2AD antigen size.The growth characteristics of rPRV-E2AD and parent strains were basically the same in ST,IPEC-J2,PK-15 and Vero cells,both of which caused cell fusion at 12 h after infection and cell loss at 36 h after infection.Physical and chemical characteristics test results showed that the recombinant strain and the parent strain had similar culture characteristics under the same physical and chemical conditions.【Conclusion】 In this study,recombinant virus rPRV-E2AD was successfully obtained,and the foreign gene did not affect the proliferation characteristics of the parent strain.The results provided the candidate strains for the development of recombinant CSFV E2 gene live vector vaccine,and also provided the research basis for the development of PRV live vector vaccine.

Key words: Classical swine fever virus (CSFV); Pseudorabies virus (PRV); recombinant virus; E2 gene

中图分类号:

S852.65⁺1

王林青, 张刘辉, 陈曦艋, 马世杰, 宋月, 陈红英. 猪瘟病毒E2基因部分表位区重组猪伪狂犬病病毒的构建及生物学特性研究[J]. 中国畜牧兽医, 2025, 52(4): 1815-1824.

WANG Linqing, ZHANG Liuhui, CHEN Ximeng, MA Shijie, SONG Yue, CHEN Hongying. Construction and Biological Characteristics of Recombinant PRV in Partial Epitope Region of CSFV E2 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1815-1824.

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