携带eGFP基因的重组牛结节性皮肤病病毒的构建及其生长特性研究

doi:10.16431/j.cnki.1671-7236.2024.12.034

摘要/Abstract

摘要： 【目的】筛选可高效率表达重组牛结节性皮肤病病毒(Lumpy skin disease virus,LSDV)的不同启动子/细胞组合，据此构建携带增强绿色荧光蛋白(eGFP)基因的重组LSDV，并探究其生长特性,为后续抗病毒药物的开发以及病毒复制和作用机制的研究奠定基础。【方法】利用融合PCR方法分别将羊口疮病毒(Orf virus,ORFV)启动子、LSDV启动子与eGFP基因进行连接，用以替换LSDV 005基因，构建同源重组片段，并将其分别转染至感染LSDV的非洲绿猴肾细胞(Vero)、非洲绿猴肾细胞衍生株(Vero-E6)、牛肾细胞(MDBK)、仓鼠卵巢细胞(CHO-K1)、羊睾丸细胞(LT)和原代羊睾丸细胞(PLT)6种细胞中，通过荧光显微镜观察转染效果，并使用ImageJ软件统计荧光面积百分比,筛选出最优启动子/细胞组合。以筛选出的最优组合进行重组病毒转染，并检测病毒纯化效果，进一步探究重组病毒的生长特性和稳定性。【结果】在6种细胞中LSDV启动子的转染效果均优于ORFV启动子，且在PLT细胞中，LSDV启动子的荧光面积百分比最高(8.38%)，与其他组细胞相比差异显著(P＜0.05)。使用LSDV启动子/PLT细胞组合构建重组病毒LSDV-Δ005/eGFP，纯化后在倒置荧光显微镜下观察到所有病变部位均带有绿色荧光，荧光面积百分比约为50.8%。病毒的生长特性和稳定性分析结果显示，重组病毒LSDV-Δ005/eGFP在6种细胞中的病变效应以及病毒滴度随时间变化的趋势与野生毒株LSDV/Heilongjiang/2022相似，且在PLT细胞上连续传代10次eGFP基因稳定表达。【结论】利用LSDV启动子/PLT细胞组合构建重组LSDV时效率最佳，据此构建的重组病毒LSDV-Δ005/eGFP生长特性与LSDV野生毒株一致，经10次传代后遗传稳定性良好。

关键词: 牛结节性皮肤病病毒; 细胞转染; 重组病毒; 生长特性

Abstract: 【Objective】 The aim of this experiment was to screen different promoter/cell combinations that could efficiently express recombinant Lumpy skin disease virus (LSDV),and thus construct recombinant LSDV carrying enhanced green fluorescent protein (eGFP) gene.The growth characteristics of the virus were studied,which laid the foundation for the development of antiviral drugs and the study of viral replication and mechanism of action.【Method】 The Orf virus (ORFV) promoter and LSDV promoter were linked to eGFP gene by fusion PCR to replace LSDV 005 gene and construct homologous recombinant fragment，and transfect it into African green monkey kidney cells (Vero),African green monkey kidney cell derived line (Vero-E6)，Madin-Darby bovine kidney cell (MDBK),hamster ovary cells (CHO-K1),lamp testicular cells (LT),and primary lamp testicular cells (PLT) infected with LSDV,respectively.The transfection effect was observed by fluorescence microscopy,and the optimal promoter/cell combination was selected by using ImageJ software to calculate the percentage of fluorescence area.The recombinant virus was transfected with the optimal combination,and the purification effect of the virus was tested.The growth characteristics and stability of recombinant virus were further studied.【Result】 The transfection effect of LSDV promoter was superior to that of ORFV promoter in all 6 kinds of cells,and the fluorescence area percentage of LSDV promoter in PLT cells was the highest (8.38%),which was significantly different from that of other groups (P ＜ 0.05).The recombinant virus LSDV-Δ005/eGFP was constructed using the combination of LSDV promoter/PLT cells.After purification,green fluorescence was observed in all the lesion sites under inverted fluorescence microscope,and the fluorescence area percentage was about 50.8%.The growth characteristics and stability analysis of the virus showed that the pathological effect of the recombinant virus LSDV-Δ005/eGFP in 6 kinds of cells and the trend of viral titer change over time was similar to that of the wild virus LSDV/Heilongjiang/2022,and the eGFP gene was statically expressed in PLT cells after 10 consecutive generations.【Conclusion】 The recombinant LSDV was constructed using the combination of LSDV promoter/PLT cells,and the growth characteristics of the recombinant LSDV-Δ005/eGFP were consistent with those of wild LSDV,and the genetic stability was good after 10 passages.

Key words: Lumpy skin disease virus; cell transfection; recombinant virus; growth characteristics

中图分类号:

S852.65⁺3

莘若兰, 周祉玉, 张嘉雯, 杜吉革, 印春生, 王凤雪, 陈小云, 温永俊, 朱真. 携带eGFP基因的重组牛结节性皮肤病病毒的构建及其生长特性研究[J]. 中国畜牧兽医, 2024, 51(12): 5479-5488.

XIN Ruolan, ZHOU Zhiyu, ZHANG Jiawen, DU Jige, YIN Chunsheng, WANG Fengxue, CHEN Xiaoyun, WEN Yongjun, ZHU Zhen. Construction and Growth Characteristics of Recombinant Lumpy Skin Disease Virus Carrying eGFP Gene[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5479-5488.

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