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

Abstract

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

CLC Number:

S852.65⁺3

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.

References

[1] 谢士杰, 朱俊达, 王天坤, 等.牛结节性皮肤病疫苗研究进展[J].中国畜牧兽医, 2021, 48(11):4220-4230.XIE S J, ZHU J D, WANG T K, et al.Research progress on vaccine development for lumpy skin disease[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(11):4220-4230.(in Chinese)
[2] AKTHER M, AKTER S H, SARKER S, et al.Global burden of lumpy skin disease, outbreaks, and future challenges[J].Viruses, 2023, 15(9):1861
[3] 鲁志平, 汪熙来, 李燕, 等.牛结节性皮肤病及综合防控措施分析[J].山东畜牧兽医, 2024, 45(3):37-38.LU Z P, WANG X L, LI Y, et al.Analysis of lumpy skin disease and comprehensive preventive and control measures[J].Shandong Journal of Animal Science and Veterinary Medicine, 2024, 45(3):37-38.(in Chinese)
[4] 张辉, 张慧, 余翰维, 等.牛结节性皮肤病诊断方法研究进展[J].畜牧与兽医, 2023, 55(7):138-144.ZHANG H, ZHANG H, YU H W, et al.Advances in researches on methods for diagnosing lumpy skin diseases[J].Animal Husbandry & Veterinary Medicine，2023，55(7):138-144.(in Chinese)
[5] KONONOVA S, KONONOV A, SHUMILOVA I, et al.A Lumpy skin disease virus which underwent a recombination event demonstrates more aggressive growth in primary cells and cattle than the classical field isolate[J].Transboundary and Emerging Diseases, 2021, 68(3):1377-1383.
[6] VAN DIEPEN M, CHAPMAN R, DOUGLASS N, et al.Advancements in the growth and construction of recombinant Lumpy skin disease virus (LSDV) for use as a vaccine vector[J].Vaccines-Basel，2021，9(10):1131.
[7] MA J, YUAN Y, SHAO J, et al.Genomic characterization of Lumpy skin disease virus in Southern China[J].Transboundary and Emerging Diseases，2022, 69(5):2788-2799.
[8] 聂福平.牛结节性皮肤病检测新方法与ORF132基因表达及鉴定研究[D].重庆：重庆大学，2022.NIE F P.Research of molecular methods for the detection of lumpy skin disease and the expression of ORF132 gene in Baculovirus system and identification[D].Chongqing:Chongqing University, 2022.(in Chinese)
[9] BALINSKY C A, DELHON G, AFONSO C L, et al.Sheeppox virus kelch-like gene SPPV-019 affects virus virulence[J].Journal of Virology，2007，81(20):11392-11401.
[10] KARA P D, MATHER A S, PRETORIUS A, et al.Characterisation of putative immunomodulatory gene knockouts of Lumpy skin disease virus in cattle towards an improved vaccine[J].Vaccine, 2018, 36(31):4708-4715.
[11] CHERVYAKOVA O，ISSABEK A，SULTANKULOVA K，et al.Lumpy skin disease virus with four knocked out genes was attenuated in vivo and protects cattle from infection[J].Vaccines-Basel，2022，10(10):1705.
[12] GOPINATH K, WARNER D F, MIZRAHI V.Targeted gene knockout and essentiality testing by homologous recombination[J]. Methods in Molecular Biology, 2015，1285:131-149.
[13] 彭金美, 王瑜, 田志军, 等.带BAC质粒的重组伪狂犬病毒的构建及其体外生长特性研究[J].中国预防兽医学报，2009, 31(1):10-15.PENG J M, WANG Y, TIAN Z J, et al.Construction of recombinant Pseudorabies virus with BAC plasmid and its in vitro growth characteristics[J].Chinese Journal of Preventive Veterinary Medicine, 2009，31(1):10-15.(in Chinese)
[14] 丁海峰, 李小申, 黄慧, 等.基因敲除技术研究进展[J].黑龙江畜牧兽医, 2018, 5:50-54.DING H F, LI X S, HUANG H, et al.The research progress of gene knockout technology[J].Heilongjiang Animal Science and Veterinary Medicine, 2018, 5:50-54.(in Chinese)
[15] 赵广伟, 杨小玲, 张孟奇, 等.不同转染方法应用于体细胞转基因效率的研究[J].家畜生态学报, 2024, 45(2):13-18.ZHAO G W, YANG X L, ZHANG M Q, et al.Studies on the efficiency of different transfection methods applied to somatic cell transgenesis[J].Journal of Domestic Animal Ecology, 2024, 45(2):13-18.(in Chinese)
[16] 肖梦珂, 华宇, 冯莹莹, 等.不同核心启动子对中国仓鼠卵巢细胞重组蛋白表达的影响[J].新乡医学院学报, 2021, 38(6):501-504.XIAO M K, HUA Y, FENG Y Y, et al.Effects of different core promoters on recombinant protein expression in Chinese hamster ovary cells[J].Journal of Xinxiang Medical College, 2021, 38(6):501-504.(in Chinese)
[17] PRIDEAUX C T, KUMAR S, BOYLE D B.Comparative analysis of Vaccinia virus promoter activity in fowlpox and Vaccinia virus recombinants[J].Virus Research，1990, 16(1):43-57.
[18] FAY P C, COOK C G, WIJESIRIWARDANA N, et al.Madin-Darby bovine kidney (MDBK) cells are a suitable cellline for the propagation and study of the Bovine poxvirus Lumpy skin disease virus[J].Journal of Virological Methods，2020, 285:113943.
[19] 马小琴.牛结节性皮肤病病毒基因缺失毒株的构建及生物学特性分析[D].北京：中国农业科学院, 2023.MA X Q.Construction and biological characteristics analysis of a gene deletion strain of Lumpy skin disease virus[D].Beijing:Chinese Academy of Agricultural Sciences, 2023.(in Chinese)
[20] 陈勇.羊传染性脓疱病毒cbp/gif双基因缺失株的构建及其生物学特性研究[D].北京：中国兽医药品监察所, 2021.CHEN Y.Construction of cbp/gif double gene-deleted contagious Ecthyma virus and its biological characteristics[D].Beijing:China Institute of Veterinary Drug Control, 2021.(in Chinese)
[21] 覃鸿妮, 谢钰珍, 张楠楠, 等.小胶质细胞中不同启动子驱动外源基因表达效率比较[J/OL].首都师范大学学报(自然科学版), 1-10[2024-07-15].http://kns.cnki.net/kcms/detail/11.3189.n.20240423.1019.002.html.QIN H N, XIE Y Z, ZHANG N N, et al.Comparison of the efficiency of different promoters driving foreign gene expression in microglia[J/OL].Journal of Capital Normal University (Natural Sciences Edition)，1-10[2024-07-15].http://kns.cnki.net/kcms/detail/11.3189.n.20240423.1019.002.html. (in Chinese)
[22] 阚恩晞, 祝徐航, 张孝辉, 等.两种启动子拯救猪繁殖与呼吸综合征病毒ZJ-JX-2015株效率的比较研究[J].中国预防兽医学报, 2023, 45(5):529-534.KAN E X, ZHU X H, ZHANG X H, et al.Comparative study on the efficiency of two promoters to rescue Porcine reproductive and respiratory syndrome virus strain ZJ-JX-2015[J].Chinese Journal of Preventive Veterinary Medicine, 2023, 45(5):529-534.(in Chinese)
[23] WANG J, JI J, ZHONG Y, et al.Construction of recombinant fluorescent LSDV for high-throughput screening of antiviral drugs[J].Veterinary Research，2024, 55(1):33.

Construction and Growth Characteristics of Recombinant Lumpy Skin Disease Virus Carrying eGFP Gene

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 7

Recommended Articles

Metrics

Comments

[1]	LI Nan, MENG Jinxin, WANG Jiannie, HE Yuwen, YANG Shaochang, LYU Shunyan, WANG Jinglin. Detection of Lumpy Skin Disease Virus in Cattle and Investigation of Virus Carried by Midges and Mosquitoes [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4179-4187.
[2]	WEI Xiao, WANG Qiuju, SUN Yu, ZHANG Hao, LI Changyang, TAO Yanzi. Isolation and Identification of Bacillus tequilensis and Weissella paramesenteroides from Goose Feces [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2778-2787.
[3]	SHANG Yu, LI Lintao, LI Li, XU Yingying, FENG Helong, WANG Hongcai, ZHANG Rongrong, ZENG Zhe, WANG Honglin, LUO Qingping, SHAO Huabin, WEN Guoyuan. Construction of Recombinant Heat-resistant Newcastle Disease Virus Expressing Fiber2 Protein of Fowl Adenovirus Serotype 4 and Evaluation of Its Immune Effect [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 650-659.
[4]	YIN Juanbin, ZHOU Yahua, YIN Xiangping, ZHANG Zhidong. Rescue of Recombinant Feline Panleukopenia Virus Carrying GFP Gene [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1853-1860.
[5]	YANG Chun, ZHU Hong-wei, LIU Zong-yue, WANG Gui-wu, YANG Fu-he, XING Xiu-mei. Establishment of Liver Specific Expression of Cre Recombinase Transgenic Pig Fibroblast [J]. , 2012, 39(10): 145-149.
[6]	GAO Pei, MA Yu-zhen, YIN Jun, ZHOU Huan-min. Influences of FGF5 Gene Knockout to Arbas Cashmere Goat Fetal Skin Fibroblast Cell Line [J]. , 2012, 39(10): 27-32.
[7]	ZHU Xue-liang;;ZHANG Qiang;YANG Fan;;CAI Xue-peng. Progress of Research on the Vector of Capripoxvirus [J]. , 2009, 36(5): 98-101.