非洲猪瘟病毒<em>C129R</em>基因双标签重组病毒的构建与鉴定

doi:10.3969/j.issn.1674-7968.2026.10.017

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摘要非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引起的高致死性烈性传染病,对全球养猪业造成毁灭性损失,目前尚无安全有效的商品化疫苗,解析病毒关键蛋白功能是疫苗研发与抗病毒策略设计的核心前提。传统ASFV蛋白功能研究高度依赖特异性抗体,存在抗体制备周期长、流程繁杂等局限。C129R是ASFV核心区域编码的结构蛋白,其在病毒颗粒中的精确定位、动态分布及宿主互作机制尚未明确。为研究C129R在病毒感染与致病中的功能,本研究构建非洲猪瘟病毒结构蛋白C129R的羧基端3×Flag-Strep双标签重组毒株,利用同源重组技术,将编码3×Flag-Strep双标签及GFP筛选标记的基因序列插入ASFV CN/GS/2018毒株C129R基因羧基端,通过克隆筛选纯化获得融合重组病毒ASFV-C129R-3×Flag-Strep。Western blot检测证实重组病毒正确表达携带双标签的C129R融合蛋白;qPCR显示,标签插入未显著影响C129R及其上下游基因M448R和C84L的转录水平;一步生长曲线、血细胞吸附试验及透射电镜观察表明,重组病毒与亲本毒株的体外复制动力学、形态结构及病毒装配均无显著差异。本研究成功构建了生物学特性稳定的C129R双标签重组ASFV,为后续解析C129R在病毒颗粒中的精确定位、结构特征及其与宿主蛋白相互作用机制提供参考。

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	甘露露
	周艳龙
	任静静
	颜丽娇
	郑海学
	李丹

关键词 ：非洲猪瘟病毒, C129R, Strep标签, Flag标签, 重组病毒

Abstract：African swine fever (ASF) is a highly lethal infectious disease caused by African swine fever virus (ASFV), inflicting devastating losses on the global pig industry. Currently, no safe and effective commercial vaccines are available, and elucidating the functions of viral key proteins serves as the core prerequisite for vaccine development and antiviral strategy design. Traditional research on ASFV protein functions heavily relies on specific antibodies, which is limited by long preparation cycles and cumbersome procedures. C129R is a structural protein encoded by the central region of ASFV, while its precise localization in viral particles, dynamic distribution and host interaction mechanisms remain unclear. To investigate the functions of C129R during viral infection and pathogenesis, in this study, a recombinant ASFV strain with a C-terminal 3×Flag-Strep double tag fused to the structural protein C129R was constructed. Using homologous recombination technology, the gene sequences encoding the 3×Flag-Strep double tag and GFP selection marker were inserted into the C-terminus of the C129R gene of ASFV CN/GS/2018 strain, and the recombinant virus ASFV-C129R-3×Flag-Strep was obtained through clonal screening and purification. Western blot analysis confirmed that the recombinant virus correctly expressed the C129R fusion protein carrying the double tag. qPCR revealed that tag insertion had no significant effect on the transcription levels of C129R and its upstream and downstream genes M448R and C84L. One-step growth curve, hemadsorption assay and transmission electron microscopy showed no significant differences in in vitro replication kinetics, morphological structure and viral assembly between the recombinant virus and the parental strain.This study successfully constructed a double-tagged recombinant ASFV targeting C129R with stable biological characteristics, providing a technical reference for further analyzing the precise localization and structural features of C129R in viral particles, as well as its interaction mechanism with host proteins.

Key words： African swine fever virus (ASFV) C129R Strep tag Flag tag Recombinant virus

收稿日期: 2026-02-03

中图分类号： S852.65

基金资助:甘肃省基础研究创新群体项目(25JRRA434); 甘肃省陇原青年英才项目(20230102); 甘肃省重大科技项目(22ZD6NA001); 甘肃省重点人才项目(212068657016)

通讯作者: ^*lidan@caas.cn;zhenghaixue@caas.cn

引用本文:

甘露露, 周艳龙, 任静静, 颜丽娇, 郑海学, 李丹. 非洲猪瘟病毒C129R基因双标签重组病毒的构建与鉴定[J]. 农业生物技术学报, 2026, 34(10): 2272-2280.
GAN Lu-Lu, ZHOU Yan-Long, REN Jing-Jing, YAN Li-Jiao, ZHENG Hai-Xue, LI Dan. Construction and Identification of a C129R Gene Double-tagged Recombinant African swine fever virus. 农业生物技术学报, 2026, 34(10): 2272-2280.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2026.10.017 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2026/V34/I10/2272

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