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牛结节性皮肤病病毒（Lumpy skin disease virus, LSDV）引起的牛结节性皮肤病（Lumpy skin disease, LSD）是一种牛全身性感染疫病，对养牛产业的健康发展造成了严重威胁。本研究旨在通过构建缺失ORF143基因重组LSDV毒株，并进行其生物学特性分析，为LSDV致弱毒株的筛选及弱毒疫苗研发提供候选毒株。首先，利用同源重组技术，构建pUC19T-LSDV-ΔORF143-EGFP转移载体，再将此转移载体转染至Vero细胞并感染LSDV，利用有限稀释与单细胞亚克隆方法筛选出纯合的以增强型绿色荧光蛋白（EGFP）为标识物的ORF143基因缺失重组LSDV(rLSDV-ΔORF143-EGFP)。对该重组病毒的生长曲线、滴度和遗传稳定性等进行分析。PCR扩增与重组病毒测序结果表明该rLSDV-ΔORF143-EGFP毒株已纯化成功，且至少20代内可稳定遗传；生长曲线结果表明该重组毒株增殖能力低于野生型LSDV毒株（LSDV-WT）。本研究成功构建并纯化出重组病毒rLSDV-ΔORF143-EGFP，为LSDV弱毒疫苗研究提供候选毒株，为LSD新型疫苗研究提供理论基础。
Abstract：Lumpy skin disease(LSD), caused by lumpy skin disease virus(LSDV), is a systemic infection disease of cattle that poses a serious threat to the sustainable development of the cattle industry. This study aimed to construct an ORF143 gene-deleted recombinant LSDV strain and evaluate its biological characteristics, in order to provide a candidate strain for the development of gene-deleted attenuated vaccines against LSDV. A transfer vector, pUC19T-LSDV-ΔORF143-EGFP, was constructed using homologous recombination technology. This vector was transfected into Vero cells, which were then infected with LSDV.Through limited dilution and single-cell subcloning, a purified recombinant LSDV strain with deletion of the ORF143 gene and expression of enhanced green fluorescent protein(EGFP) as a reporter marker(rLSDV-ΔORF143-EGFP) was successfully obtained. The recombinant virus was characterized for its growth kinetics, viral titers, and genetic stability. PCR amplification and sequencing confirmed the successful purification of the recombinant strain, which remained genetically stable over at least 20 serial passages. Growth curve analysis revealed that rLSDV-ΔORF143-EGFP had a reduced replication capacity compared to the wild-type LSDV strain(LSDV-WT). In conclusion, this study successfully constructed and purified the recombinant strain rLSDV-ΔORF143-EGFP, which represents a promising candidate for the development of attenuated LSDV vaccines and provides a theoretical foundation for the advancement of novel LSD vaccine strategies.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250040
中图分类号:S852.65
引用信息:
[1]李家琪,柯群华,黄伟涛,等.LSDV-ORF143基因缺失重组病毒毒株的构建及其生物学特性分析[J].病毒学报,2025,41(04):1056-1066.DOI:10.13242/j.cnki.bingduxuebao.250040.
基金信息:
甘肃省联合基金项目（项目号：23JRRA1481）,题目：牛结节性皮肤病等反刍动物疫病新型疫苗研发~~