Abstract：
Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Genetic manipulation of the LSDV is essential for the elucidation of the pathogenic mechanism and biological function of the LSDV-encoded protein. In this study, we established a platform for the Cre-loxP recombination system under a modified early-late H5 promoter of the VACV for quick construction of the recombinant LSDV virus. The recombinant virus, LSDV-EGFP-ΔTK, was purified and obtained using serial limited dilution and picking the single cells methods. Using the lentiviral package system, a Cre recombinase enzyme stable expression MDBK cell line was established to supply the Cre recombinase for the reporter gene excision. A genetically stable, safe TK gene-deleted LSDV (LSDV-ΔTK) was constructed using homologous recombination and the Cre-loxP system. It was purified using limited dilution in the MDBK-Cre cell line. Establishing the Cre-loxP recombination system will enable sequential deletion of the interested genes from the LSDV genome and genetic manipulation of the LSDV genome, providing technical support and a platform for developing the attenuated LSDV vaccine.
摘要：
结节性皮肤病病毒(LSDV)是一种在亚洲迅速崛起的病原体,包括中国。LSDV的遗传操作对于阐明LSDV编码蛋白的致病机制和生物学功能至关重要。在这项研究中,我们在VACV的改良的早晚期H5启动子下建立了Cre-loxP重组系统的平台，以快速构建重组LSDV病毒。重组病毒，LSDV-EGFP-ΔTK,使用连续有限稀释和挑选单细胞方法纯化和获得。使用慢病毒包装系统，建立Cre重组酶稳定表达的MDBK细胞系以提供用于报告基因切除的Cre重组酶。一个基因稳定的，使用同源重组和Cre-loxP系统构建了安全的TK基因缺失的LSDV（LSDV-ΔTK）。在MDBK-Cre细胞系中使用有限稀释对其进行纯化。建立Cre-loxP重组系统将能够从LSDV基因组中顺序删除感兴趣的基因，并对LSDV基因组进行遗传操作。为开发减毒LSDV疫苗提供技术支持和平台。
