牛结节性皮肤病病毒ORF123基因缺失重组病毒的构建及增殖能力分析

doi:10.11843/j.issn.0366-6964.2024.10.024

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4530-4541.doi: 10.11843/j.issn.0366-6964.2024.10.024

牛结节性皮肤病病毒ORF123基因缺失重组病毒的构建及增殖能力分析

王芳萍¹(), 任善会²^,*(), 郜晓虹², 杨雪¹, 李积雲³, 王相伟², 殷相平², 孙跃峰², 陈豪泰², 万学瑞¹^,*()

1. 甘肃农业大学动物医学院, 兰州 730070
2. 中国农业科学院兰州兽医研究所, 兰州 730046
3. 青海省科学技术信息研究所, 西宁 810003

收稿日期:2023-10-18 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 任善会,万学瑞 E-mail:344884873@qq.com;renshanhui@caas.cn;wanxr@gsau.edu.cn
作者简介:王芳萍(1998-), 女, 甘肃天水人, 硕士生, 主要从事兽医微生物与免疫学研究, E-mail: 344884873@qq.com
基金资助:
国家自然科学基金(32302850);甘肃省科技计划资助(22JR5RA035);甘肃省科技重大专项(22ZD6NA001);中国农业科学院兰州兽医研究所基本科研业务费(1610312021008)

Construction and Growth Characteristics of ORF123 Deleted Lumpy Skin Disease Virus Strain

Fangping WANG¹(), Shanhui REN²^,*(), Xiaohong GAO², Xue YANG¹, Jiyun LI³, Xiangwei WANG², Xiangping YIN², Yuefeng SUN², Haotai CHEN², Xuerui WAN¹^,*()

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
3. Qinghai Academy of Science and Technology, Xining 810003, China

Received:2023-10-18 Online:2024-10-23 Published:2024-11-04
Contact: Shanhui REN, Xuerui WAN E-mail:344884873@qq.com;renshanhui@caas.cn;wanxr@gsau.edu.cn

摘要/Abstract

摘要：

旨在通过缺失牛结节性皮肤病病毒(LSDV)ORF123基因，分析其增殖能力，为LSDV ORF123功能研究奠定基础。本研究利用同源重组，以ORF123基因作为靶标，增强型绿色荧光蛋白(EGFP)为筛选标记，采用融合PCR方法，扩增同源左右臂序列以及EGFP基因表达框，克隆至pUC-19T载体，构建基因缺失转移载体pH5-LSDVΔORF123-EGFP质粒。然后将pH5-LSDVΔORF123-EGFP质粒转染至Vero细胞，并感染LSDV/CHA/FJ/2021毒株，利用蚀斑、梯度稀释和挑取绿色荧光单克隆细胞筛选纯化重组毒株，并鉴定其遗传稳定性和增殖能力。结果显示，利用EGFP筛选标记，通过多次挑斑筛选纯化获得ORF123基因缺失的重组病毒株LSDVΔORF123-EGFP。该重组病毒至少在8代细胞传代中能稳定表达绿色荧光蛋白，接种MDBK细胞后绘制一步增殖曲线表明该重组病毒滴度略低于亲本毒株。本研究成功获得ORF123基因缺失的重组病毒株LSDVΔORF123-EGFP，为LSDV ORF123蛋白生物学功能研究以及减毒活疫苗的研制奠定基础。

关键词: 牛结节性皮肤病病毒, ORF123基因, 增殖能力, 同源重组, 重组病毒

Abstract:

The aim of this study was to analyze the growth characteristics of the Lumpy skin disease virus (LSDV) strain with the ORF123 gene deleted, which laid a foundation for the functional research of LSDV ORF123. ORF123 was used as the target gene and enhanced green fluorescent protein (EGFP)was used as the screening marker, the homologous left and right arm sequences and the EGFP gene expression frame were amplified and fused by overlapping PCR and cloned into pUC-19T vector to construct pH5-LSDVΔORF123-EGFP. The plasmid pH5-LSDVΔORF123-EGFP was then transfected into Vero cells and after that the cells were infected with the LSDV/CHA/FJ/2021 strain. The recombinant virus strain LSDVΔORF123-EGFP was purified through multiple plaque screening using EGFP as a screening markers, and its viral genetic stability and growth characteristics were characterized. The results revealed that the recombinant virus was able to stably express green fluorescent protein across at least 8 cell passages and the one-step growth curve following MDBK cell inoculation indicated that the titer of the recombinant virus was slightly lower than that of the parental strain. In conclusion, the recombinant virus strain LSDVΔORF123-EGFP was successfully obtained, which laid a foundation for the study of the biological function of LSDV ORF123 protein and the development of a live attenuated LSDV vaccine.

Key words: lumpy skin disease virus, ORF123 gene, growth characteristic, homologous recombination, recombinant virus

中图分类号:

S852.654

王芳萍, 任善会, 郜晓虹, 杨雪, 李积雲, 王相伟, 殷相平, 孙跃峰, 陈豪泰, 万学瑞. 牛结节性皮肤病病毒ORF123基因缺失重组病毒的构建及增殖能力分析[J]. 畜牧兽医学报, 2024, 55(10): 4530-4541.

Fangping WANG, Shanhui REN, Xiaohong GAO, Xue YANG, Jiyun LI, Xiangwei WANG, Xiangping YIN, Yuefeng SUN, Haotai CHEN, Xuerui WAN. Construction and Growth Characteristics of ORF123 Deleted Lumpy Skin Disease Virus Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4530-4541.

图/表 11

表 1

图 1

图 2

图 3

图 4

图 5

图 6

图 7

图 8

图 9

图 10

参考文献 27

1	TUPPURAINEN E S M , OURA C A L . Review: lumpy skin disease: an emerging threat to Europe, the middle East and Asia[J]. Transbound Emerg Dis, 2012, 59 (1): 40- 48. doi: 10.1111/j.1865-1682.2011.01242.x
2	NAMAZI F , KHODAKARAM TAFTI A . Lumpy skin disease, an emerging transboundary viral disease: a review[J]. Vet Med Sci, 2021, 7 (3): 888- 896. doi: 10.1002/vms3.434
3	周雪梅. 牛疙瘩皮肤病毒ORF132的克隆、表达及生物信息学分析[D]. 重庆: 西南大学, 2012.
	ZHOU X M. The cloning, expression and bioinformatics analysis of ORF132 gene of Lumpy skin disease virus[D]. Chongqing: Southwest University, 2012. (in Chinese)
4	AERTS L , HAEGEMAN A , DE LEEUW I , et al. Detection of clinical and subclinical lumpy skin disease using ear notch testing and skin biopsies[J]. Microorganisms, 2021, 9 (10): 2171. doi: 10.3390/microorganisms9102171
5	WHITTLE L , CHAPMAN R , WILLIAMSON A L . Lumpy skin disease-an emerging cattle disease in Europe and Asia[J]. Vaccines (Basel), 2023, 11 (3): 578. doi: 10.3390/vaccines11030578
6	刘平, 李金明, 陈荣贵, 等. 我国首例牛结节性皮肤病的紧急流行病学调查[J]. 中国动物检疫, 2020, 37 (1): 1- 5.
	LIU P , LI J M , CHEN R G , et al. The first outbreak investigation of lumpy skin disease in China[J]. China Animal Health Inspection, 2020, 37 (1): 1- 5.
7	ARJKUMPA O , SUWANNABOON M , BOONRAWD M , et al. First emergence of lumpy skin disease in cattle in Thailand, 2021[J]. Transbound Emerg Dis, 2021, 68 (6): 3002- 3004. doi: 10.1111/tbed.14246
8	BHANUPRAKASH V , HOSAMANI M , VENKATESAN G , et al. Animal poxvirus vaccines: a comprehensive review[J]. Expert Rev Vaccines, 2012, 11 (11): 1355- 1374. doi: 10.1586/erv.12.116
9	KHAN Y R , ALI A , HUSSAIN K , et al. A review: surveillance of lumpy skin disease (LSD) a growing problem in Asia[J]. Microb Pathog, 2021, 158, 105050. doi: 10.1016/j.micpath.2021.105050
10	TULMAN E R , AFONSO C L , LU Z , et al. Genome of lumpy skin disease virus[J]. J Virol, 2001, 75 (15): 7122- 7130. doi: 10.1128/JVI.75.15.7122-7130.2001
11	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, 2020.
	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, 2020. (in Chinese)
12	LIANG Z J , WANG S S , YAO K S , et al. Lumpy skin disease virus ORF127 protein suppresses type Ⅰ interferon responses by inhibiting K63-linked ubiquitination of tank binding kinase 1[J]. FASEB J, 2024, 38 (3): e23467. doi: 10.1096/fj.202301987RR
13	HOOPER J W , CUSTER D M , SCHMALJOHN C S , et al. DNA vaccination with vaccinia virus L1R and A33R genes protects mice against a lethal poxvirus challenge[J]. Virology, 2000, 266 (2): 329- 339. doi: 10.1006/viro.1999.0096
14	ROPER R L , PAYNE L G , MOSS B . Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene[J]. J Virol, 1996, 70 (6): 3753- 3762. doi: 10.1128/jvi.70.6.3753-3762.1996
15	ROPER R L , WOLFFE E J , WEISBERG A , et al. The envelope protein encoded by the A33R gene is required for formation of actin-containing microvilli and efficient cell-to-cell spread of vaccinia virus[J]. J Virol, 1998, 72 (5): 4192- 4204. doi: 10.1128/JVI.72.5.4192-4204.1998
16	SU H P , SINGH K , GITTIS A G , et al. The structure of the poxvirus A33 protein reveals a dimer of unique C-type lectin-like domains[J]. J Virol, 2010, 84 (5): 2502- 2510. doi: 10.1128/JVI.02247-09
17	王丹, 张新玉, 付佳棋, 等. 绵羊SMAD4基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50 (12): 4783- 4792.
	WANG D , ZHANG X Y , FU J Q , et al. Cloning, Bioinformatics analysis and tissue expression of SMAD4 gene in sheep[J]. China Animal Husbandry & Veterinary Medicine, 2023, 50 (12): 4783- 4792.
18	MATSIELA M S , NAICKER L , DIBAKWANE V S , et al. Improved safety profile of inactivated neethling strain of the lumpy skin disease vaccine[J]. Vaccine X, 2022, 12, 100209. doi: 10.1016/j.jvacx.2022.100209
19	TUPPURAINEN E S M , VENTER E H , SHISLER J L , et al. Review: apripoxvirus Diseases: current status and opportunities for control[J]. Transbound Emerg Dis, 2017, 64 (3): 729- 745. doi: 10.1111/tbed.12444
20	SPRYGIN A , PESTOVA Y , BJADOVSKAYA O , et al. Evidence of recombination of vaccine strains of lumpy skin disease virus with field strains, causing disease[J]. PLoS One, 2020, 15 (5): e0232584. doi: 10.1371/journal.pone.0232584
21	TEFFERA M , BABIUK S . Potential of using capripoxvirus vectored vaccines against arboviruses in sheep, goats, and cattle[J]. Front Vet Sci, 2019, 6, 450. doi: 10.3389/fvets.2019.00450
22	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.
23	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. doi: 10.1016/j.vaccine.2018.06.017
24	BOSHRA H , TRUONG T , NFON C , et al. A lumpy skin disease virus deficient of an IL-10 gene homologue provides protective immunity against virulent capripoxvirus challenge in sheep and goats[J]. Antiviral Res, 2015, 123, 39- 49. doi: 10.1016/j.antiviral.2015.08.016
25	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. doi: 10.3390/vaccines10101705
26	陈国梁, 白兴俊, 赵蓉, 等. PCR构建融合基因方法的建立[J]. 生物技术通报, 2010, 26 (8): 209- 213.
	CHEN G L , BAI X J , ZHAO R , et al. Establishment and optimization of PCR methods for constructing fusion gene[J]. Biotechnology Bulletin, 2010, 26 (8): 209- 213.
27	XU Z Y , ZIKOS D , TAMOŠIūNAIT? A , et al. Identification of 10 cowpox virus proteins that are necessary for induction of hemorrhagic lesions (red pocks) on chorioallantoic membranes[J]. J Virol, 2014, 88 (15): 8615- 8628. doi: 10.1128/JVI.00901-14

引物名称 Primers name	引物序列(5′→3′) Primer sequence(5′→3′)	产物长度/bp Product length
ORF123L-F	CAGGTCGACTCTAGA$\underline{{\rm{GGATCC}}}$GGATACTTGGATCAACTATATGTCTAATTACAG	719
ORF123L-R	GATGAATAGGTTTAAGGAAAAACATTTTTTAAGAAATAGTTTAATAACAAATCCATTTAG	719
EGFP-F	GTTTTTCCTTAAACCTATTCATCATATAACTTCGTATGCAACATTATACGAAGTTATAAAAATTGAAAATAAATACAAAG	1 159
EGFP-R	GTAATATAATAATTTATTTTTTTTTATAACTTCGTATAATGTATGCTATACGAAGTTATTAAGATACATTGATGAGTTTG	1 159
ORF123R-F	TATAAAAAAAAATAAATTATTATATTACTATAAATGAAGTCATTAAATAGACAAACC	763
ORF123R-R	AGTGAATTCGAGCTC$\underline{{\rm{GGTACC}}}$TATTTAAAGATACTACGTTATAAGGTCCTAATGTTTC	763

[1]	杨坤, 马静文, 周新瑞, 罗烈柱, 刘喆, 胡自强, 武星辰, 梁立滨, 高诗敏. 三株传染性法氏囊病病毒重组毒株的致病性研究[J]. 畜牧兽医学报, 2024, 55(6): 2550-2559.
[2]	马春玲, 任善会, 杨雪, 蔺玉刚, 李积雲, 王相伟, 殷相平, 孙跃峰, 万学瑞, 陈豪泰. 基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立[J]. 畜牧兽医学报, 2024, 55(4): 1800-1809.
[3]	张玉哲, 任善会, 姚威, 龚真莉, 张红强, 柳民意, 尤婷, 王相伟, 李积雲, 殷相平, 孙跃峰, 陈豪泰, 万学瑞. 应用Cre-LoxP系统构建牛结节性皮肤病病毒缺失TK基因毒株[J]. 畜牧兽医学报, 2024, 55(10): 4517-4529.
[4]	张婷, 冯涛, 杨金柯, 郝雨, 杨行, 张大俊, 史喜绢, 闫文倩, 陈玲玲, 刘湘涛, 郑海学, 张克山. 条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性[J]. 畜牧兽医学报, 2023, 54(2): 706-714.
[5]	曹莉, 程如楠, 武周慧, 王家伟, 王瑜, 张永红, 吴清民, 王真. 鼠伤寒沙门菌sapC基因缺失株的构建及生物学特性分析[J]. 畜牧兽医学报, 2022, 53(7): 2282-2289.
[6]	令狐远凤, 潘永, 杨阳, 段世宇, 张家莉, 张宝太, 杨琦. 鼠伤寒沙门菌伴侣蛋白Hfq与小RNA GcvB结合位点的初步分析[J]. 畜牧兽医学报, 2022, 53(11): 4110-4115.
[7]	曹政, 居马别克·夏拉巴依, 李春燕, 马力克·艾则孜. 牛结节性皮肤病病毒感染MDBK细胞后转录组差异表达分析[J]. 畜牧兽医学报, 2021, 52(2): 460-468.
[8]	刘存, 吕桂霞, 徐鸿, 党安坤, 梁琳, 陈静, 孙圣福, 兰邹然. 一起境外输入性牛结节性皮肤病疫情[J]. 畜牧兽医学报, 2021, 52(11): 3317-3322.
[9]	高闪电, 王锦明, 田占成, 独军政, 张忠辉, 王建东, 康晓冬, 李有全, 关贵全, 刘光远, 罗建勋, 殷宏. 1株牛病毒性腹泻病毒分离毒株的基因组特征[J]. 畜牧兽医学报, 2020, 51(8): 1913-1922.
[10]	朱可蒙, 谨瑾, 王宁, 辛九庆, 刘恒贵. 蛋白质硫辛酸修饰相关酶大肠杆菌研究模型的构建[J]. 畜牧兽医学报, 2019, 50(5): 1073-1081.
[11]	周忠文, 林桂芳, 王好好, 苏帅, 崔治中, 孙淑红. 表达禽网状内皮组织增生病病毒env基因的重组马立克病毒的生物学特性[J]. 畜牧兽医学报, 2018, 49(7): 1482-1491.
[12]	李国玲, 王豪强, 阮晓芳, 莫健新, 全绒, 钟翠丽, 李紫聪, 吴珍芳, 刘德武, 张献伟. 三种小分子化合物对猪胎儿成纤维细胞DNA精确修复效率的影响[J]. 畜牧兽医学报, 2018, 49(12): 2584-2592.
[13]	王晓燕, 刘文超, 付礼胜, 高雪丽, 刘超男, 吕晓萍, 郑世民. 禽网状内皮组织增生病病毒感染SPF雏鸡血液淋巴细胞增殖功能及其细胞周期相关因子变化[J]. 畜牧兽医学报, 2018, 49(1): 157-163.
[14]	尹磊，祁克宗，涂健，周秀红，刘红梅，王曼，张艳娜. 非编码小RNA （RyhB）调控禽致病性大肠杆菌毒力相关基因的分析[J]. 畜牧兽医学报, 2015, 46(8): 1409-1416.
[15]	谢堃，乔军，孟庆玲，彭叶龙，赵海龙，马玉，陈诚，才学鹏，陈创夫. 单核细胞增生李斯特菌非编码RNA rli87基因缺失株的构建、鉴定及其生长特性初步研究[J]. 畜牧兽医学报, 2015, 46(6): 998-1003.

牛结节性皮肤病病毒ORF123基因缺失重组病毒的构建及增殖能力分析

Construction and Growth Characteristics of ORF123 Deleted Lumpy Skin Disease Virus Strain

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 11

参考文献 27

相关文章 15

编辑推荐

Metrics

本文评价