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

Abstract

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

CLC Number:

S852.659.1

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

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