应用Cre-LoxP系统构建牛结节性皮肤病病毒缺失TK基因毒株

doi:10.11843/j.issn.0366-6964.2024.10.023

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4517-4529.doi: 10.11843/j.issn.0366-6964.2024.10.023

应用Cre-LoxP系统构建牛结节性皮肤病病毒缺失TK基因毒株

张玉哲¹(), 任善会², 姚威³, 龚真莉², 张红强¹, 柳民意¹, 尤婷¹, 王相伟², 李积雲⁴, 殷相平², 孙跃峰², 陈豪泰², 万学瑞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 730046
3. 重庆市万州区畜牧产业发展中心, 重庆 400000
4. 青海省科学技术信息研究所, 西宁 810003

收稿日期:2023-11-21 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 万学瑞 E-mail:1156928206@qq.com;wanxr@gsau.edu.cn
作者简介:张玉哲(1999-), 男, 甘肃宁县人, 硕士生, 主要从事草食动物病毒病感染致病机制研究, E-mail: 1156928206@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)

Application of the Cre-LoxP System to Construct the Deleted TK Gene of the Lumpy Skin Disease Virus

Yuzhe ZHANG¹(), Shanhui REN², Wei YAO³, Zhenli GONG², Hongqiang ZHANG¹, Minyi LIU¹, Ting YOU¹, Xiangwei WANG², Jiyun LI⁴, Xiangping YIN², Yuefeng SUN², Haotai CHEN², Xuerui WAN¹^,*()

1. Gansu Agricultural University, Lanzhou 730070, China
2. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
3. Animal Husbandry Industry Development Center, Wanzhou District, Chongqing 400000, China
4. Institute of Science and Technical Information of Qinghai Province, Xining 810003, China

Received:2023-11-21 Online:2024-10-23 Published:2024-11-04
Contact: Xuerui WAN E-mail:1156928206@qq.com;wanxr@gsau.edu.cn

摘要/Abstract

摘要：

利用同源重组技术及Cre-LoxP系统平台，构建牛结节性皮肤病病毒(lumpy skin disease virus, LSDV)缺失TK基因毒株，为研制出安全、高效的LSDV基因工程疫苗提供备选毒株。以TK基因为靶基因，利用Cre-LoxP系统平台，红色荧光蛋白(RFP)为筛选标记，采用重叠PCR方法扩增左、右同源臂以及RFP基因表达框并进行融合，将其克隆至pUC19T载体，构建基因缺失转移载体pUC19T-LSDV-ΔTK-RFP。将转移载体重组质粒转染至Vero细胞，并感染LSDV/CHA/FJ/2021毒株，构建LSDV-ΔTK-RFP重组病毒。采取蚀斑法、有限稀释法纯化LSDV-ΔTK-RFP。然后，利用Cre-LoxP系统，在MDBK-Cre细胞中从病毒基因组中切除RFP标签，采取蚀斑法和有限稀释法筛选并纯化LSDV-ΔTK毒株。利用PCR及测序方法对重组毒株进行鉴定，并测定其一步生长曲线。结果表明成功构建缺失TK基因的重组毒株(LSDV-ΔTK)，重组毒株复制力略低于野生毒株。应用Cre-LoxP系统构建的缺失TK基因LSDV毒株，具有良好的遗传稳定性和复制生长特性，为后续LSDV基因工程疫苗的研制提供了候选毒株，同时拓展了Cre-LoxP系统的应用范围。

关键词: 牛结节性皮肤病病毒, TK基因, Cre/LoxP系统, 重组毒株

Abstract:

A TK-deficient Lumpy skin disease virus (LSDV) strain was constructed by homologous recombination technology and the Cre-LoxP system platform to provide an alternative strain for developing a safe and efficient LSDV genetic engineering vaccine. Using the TK gene as the target gene, using the Cre-LoxP system platform and red fluorescent protein (RFP) as screening marker, the left and right homologous arms and RFP gene expression cassettes were amplified by overlapping PCR and fused, and then cloned into pUC19T vector to construct gene deletion transfer vector pUC19T-LSDVΔTK-RFP. The transfer vector recombinant plasmid was transfected into Vero cells and infected with LSDV/CHA/FJ/2021 strain to construct LSDV-ΔTK-RFP recombinant virus. LSDV-TK-RFP was screened and purified by the plaque method and limited dilution method. Then, using the Cre-LoxP system, RFP tags were cut off from the viral genome in MDBK-Cre cells, and LSDV-TK strains were screened and purified by the plaque method and limited dilution method. PCR and sequencing identified the recombinant strain, and its one-step growth curve was determined. The recombinant strain (LSDV-ΔTK) with deletion of the TK gene was successfully constructed, and the replication ability of the recombinant strain was slightly lower than that of the wild-type strain. The LSDV strain with deletion of the TK gene constructed by the Cre-LoxP system has good genetic stability and replication growth characteristics, so it can be used as an alternative strain for the development of the LSDV genetic engineering vaccine and expands the application range of the Cre-LoxP system.

Key words: lumpy skin disease virus, TK gene, Cre-LoxP system, recombinant virus

中图分类号:

S852.659.1

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.

Yuzhe ZHANG, Shanhui REN, Wei YAO, Zhenli GONG, Hongqiang ZHANG, Minyi LIU, Ting YOU, Xiangwei WANG, Jiyun LI, Xiangping YIN, Yuefeng SUN, Haotai CHEN, Xuerui WAN. Application of the Cre-LoxP System to Construct the Deleted TK Gene of the Lumpy Skin Disease Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4517-4529.

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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
ORF67L-F	CAGGTCGACTCTAGAGGATCCATCAATGAAACTTAATGGAGTTTATGGTTTTACGTATAAAAATGAACTAAG	1 000
ORF67L-R	ACCCTCAAGAACCTTTGTATTTATTTTCAATTTTTATAACTTCGTATAATGTATGCTATACGAAGTTATTTATTTTCGTGCAATATTGATAAAATGTTTAAAAATGTTGGATCTTTTC
ORF67R-F	TAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGC	1 100
ORF67R-R	AGTGAATTCGAGCTCGGTACCCAATATCCATCTTATAACTAATCCCATAGATAAAAAATGATC
RFP-F	ACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCCACCATGGTGAGCAAGGGCGAGGAGGATAAC	807
RFP-R	TGGTATGGCTGATTATGATCTAGAGTCGCGGCCGCTTTACTTGTACAGCTCGTCCATGCCGCCGG
Primer1-TK-F	CCATGTATCTGCCATATCAAC	316
Primer1-TK-R	CCCGATGAGTTCTATTTCCT
Primer2-RFP-F	TCCTGTCCCCTCAGTTCATGTACGG	439
Primer2-RFP-R	TCGTTGTGGGAGGTGATGTCCAACTTG
Primer3-F	AGAGCCGATAACATATATAGACCCTAG	726
Primer3-R	TAACTCGTGTCTGATACCCATTTTAC

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应用Cre-LoxP系统构建牛结节性皮肤病病毒缺失TK基因毒株

Application of the Cre-LoxP System to Construct the Deleted TK Gene of the Lumpy Skin Disease Virus

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