羊口疮病毒ORF112基因缺失毒株的构建及增殖能力分析

doi:10.11843/j.issn.0366-6964.2024.11.035

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (11): 5200-5210.doi: 10.11843/j.issn.0366-6964.2024.11.035

羊口疮病毒ORF112基因缺失毒株的构建及增殖能力分析

尤婷¹(), 任善会²^,*(), 王萌¹, 张红强¹, 高小龙³, 姚威⁴, 王卉¹, 杨雪¹, 马春玲², 柳民意¹, 张玉哲¹, 王金龙¹, 孙跃峰², 陈豪泰², 王桂荣¹^,*()

1. 甘肃农业大学, 兰州 730070
2. 中国农业科学院兰州兽医研究所, 兰州 730016
3. 青海大学动物科技学院, 西宁 810003
4. 重庆市万州区畜牧产业发展中心, 重庆 400000

收稿日期:2024-01-15 出版日期:2024-11-23 发布日期:2024-11-30
通讯作者: 任善会,王桂荣 E-mail:1756404726@qq.com;renshanhui@caas.cn;wangguirong@gsau.edu.cn
作者简介:尤婷(2000-), 女, 甘肃临夏人, 硕士生, 主要从事草食动物病毒病感染致病机制研究, E-mail: 1756404726@qq.com
基金资助:
国家自然科学基金(32302850);甘肃省科技计划项目(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 Replication Ability of the ORF112 Gene Deleted Orf Virus Strain

Ting YOU¹(), Shanhui REN²^,*(), Meng WANG¹, Hongqiang ZHANG¹, Xiaolong GAO³, Wei YAO⁴, Hui WANG¹, Xue YANG¹, Chunling MA², Minyi LIU¹, Yuzhe ZHANG¹, Jinlong WANG¹, Yuefeng SUN², Haotai CHEN², Guirong WANG¹^,*()

1. Gansu Agricultural University, Lanzhou 730070, China
2. Lanzhou Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, Lanzhou 730016, China
3. College of Animal Science and Technology, Qinghai University, Xining 810003, China
4. Animal Husbandry Industry Development Center, Wanzhou District, Chongqing 400000, China

Received:2024-01-15 Online:2024-11-23 Published:2024-11-30
Contact: Shanhui REN, Guirong WANG E-mail:1756404726@qq.com;renshanhui@caas.cn;wangguirong@gsau.edu.cn

摘要/Abstract

摘要：

利用同源重组技术构建羊口疮病毒(orf virus, ORFV)ORF112基因缺失毒株，为后续研制出高效、安全的ORFV基因工程疫苗提供候选毒株。以ORF112基因为靶基因，通过重叠PCR的方法将左、右同源臂及绿色荧光蛋白(EGFP)基因表达框，进行融合后与pUC19T载体连接，构建出pUC19T-ORFV-ΔORF112-EGFP基因转移载体。将转移载体重组质粒转染到Vero细胞与WT-ORFV基因组进行同源重组。采取有限稀释法和挑取单细胞克隆法，筛选并纯化出纯合的ORFV-ΔORF112-EGFP毒株，并对该基因缺失毒株遗传稳定性和增殖特性等进行研究。结果显示：利用有限稀释和挑取单细胞克隆方法，进行阳性重组病毒纯化，通过PCR验证和测序，成功地获得ORFV-ΔORF112-EGFP重组毒株。该重组毒株在系列传代过程中EGFP稳定表达且无减弱现象。该重组ORFV-ΔORF112-EGFP毒株与野生毒株的复制生长特性基本一致。成功构建并筛选得到纯化的ORFV-ΔORF112-EGFP基因缺失毒株，该毒株具有良好的遗传稳定性和复制能力，为后续ORFV基因工程疫苗的研制提供了候选毒株。

关键词: 羊口疮病毒, ORF112基因, 同源重组, 基因缺失

Abstract:

To provide a candidate strain for the development of an efficient and safe ORFV genetic engineering vaccine, the deleted ORF112 gene recombinant ORFV strain was constructed using the homologous recombination technique. Taking the ORF 112 gene as the targeted gene, the left and right homology arms and the enhanced green fluorescent protein gene (EGFP) expression frame were in-fused by the overlapping PCR method, which was connected with the pUC19T vector to construct the pUC19T-ORFV-ΔORF112-EGFP transfer vector. Subsequently, the pUC19T-ORFV-ΔORF112-EGFP transfer vector was transfected into the Vero cells to cause the homologous recombination events with the wild-type ORFV genome. The positive ORFV-ΔORF112-EGFP strain was screened and purified by limited dilution and picking a single-cell clone method. The genetic stability and replication ability of the deleted ORF112 ORFV recombinant strain were further investigated. The positive recombinant ORFV strain was purified using limited dilution and picking a single-cell clone method. After the PCR verification and sequencing identification, the recombinant ORFV-ΔORF112-EGFP strain was successfully obtained. The expression of EGFP in the recombinant virus genome was stable, with no attenuating characteristics during the serial passages. The replication and growth characteristics of this recombinant ORFV stain are the same as wild-type ORFV. The ORFV-ΔORF112-EGFP recombinant strain has been successfully constructed, purified, and screened, which maintains good genetic stability and replication ability, providing the candidate strain for the development of a genetic engineering ORFV vaccine.

Key words: orf virus, ORF112 gene, homologous recombination, gene deletion

中图分类号:

S852.659.1

尤婷, 任善会, 王萌, 张红强, 高小龙, 姚威, 王卉, 杨雪, 马春玲, 柳民意, 张玉哲, 王金龙, 孙跃峰, 陈豪泰, 王桂荣. 羊口疮病毒ORF112基因缺失毒株的构建及增殖能力分析[J]. 畜牧兽医学报, 2024, 55(11): 5200-5210.

Ting YOU, Shanhui REN, Meng WANG, Hongqiang ZHANG, Xiaolong GAO, Wei YAO, Hui WANG, Xue YANG, Chunling MA, Minyi LIU, Yuzhe ZHANG, Jinlong WANG, Yuefeng SUN, Haotai CHEN, Guirong WANG. Construction and Replication Ability of the ORF112 Gene Deleted Orf Virus Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5200-5210.

图/表 9

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引物名称 Primers Name	引物序列(5′→3′) Primer sequences	产物长度/ bp Product length	引物用途 Primers usage
ORF112L-F	CAGGTCGACTCTAGAGGATCCCATTTGGACAAATCTACGCACCTGACGAAGCTC	854	扩增ORF112L(左同源臂)
ORF112L-R	$\underline{CAATTTTTCCCATAACTTCGTATAATGTATGCTATACGA}$AGTTATCCCCCTGTCGTTGCAAGATTCCAGAACTCCTCTTTTTAGCTTTAC	854	扩增ORF112L(左同源臂)
ORF112R-F	$\underline{AATGTATCTTAGGGATAACTTCGTATAGCATACATTAT}$ACGAAGTTATGGGGCTGTTTTTATTCGGCAATATAATAGGTGATTATTGAAC	816	扩增ORF112R(右同源臂)
ORF112R-R	AGTGAATTCGAGCTCGGTACCTTGACGAGGGCGCGGGAAACCATGAGCTTCCGGAACAG	816	扩增ORF112R(右同源臂)
EGFP-F	$\underline{TCGTATAGCATACATTATACGAAGTTATGG}$GAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAG	1 112	扩增绿色荧光蛋白表达框(EGFP)
EGFP-R	$\underline{CCCATAACTTCGTATAATGTATGCTATACGAAGTTATCCCTAAGATACATTGATGAGTTTG}$GACAAACCACAACTAG	1 112	扩增绿色荧光蛋白表达框(EGFP)
Primer1-For	ACTGGCCATCCAGATAGGGT	299	验证ORF112基因
Primer1-Rev	ATTCCCCCTTCAGCGACAAC	299	验证ORF112基因
Primer2-For	TCGTATAGCATACATTATACGAAGTTATGGGAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAG	1 112	验证EGFP荧光标签
Primer2-Rev	CCCATAACTTCGTATAATGTATGCTATACGAAGTTATCCCTAAGATACATTGATGAGTTTGGACAAACCACAACTAG	1 112	验证EGFP荧光标签
Primer3-For	GTTCTCTAAGCAAGAACACTGG	1 146	验证缺失ORF112基因
Primer3-Rev	CCAGTTAAGCATATTACGGCG	1 146	验证缺失ORF112基因

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羊口疮病毒ORF112基因缺失毒株的构建及增殖能力分析

Construction and Replication Ability of the ORF112 Gene Deleted Orf Virus Strain

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