Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (10): 4530-4541.doi: 10.11843/j.issn.0366-6964.2024.10.024
• Preventive Veterinary Medicine • Previous Articles Next Articles
Fangping WANG1(
), Shanhui REN2,*(), Xiaohong GAO2, Xue YANG1, Jiyun LI3, Xiangwei WANG2, Xiangping YIN2, Yuefeng SUN2, Haotai CHEN2, Xuerui WAN1,*()
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
CLC Number:
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.
Table 1
The primer sequences used in this study"
| 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 |
Fig. 1
Schematic diagram of the construction for the LSDV strain deleted ORF123 gene"
Fig. 2
Results of fragment amplification results M. DNA Marker.DL5000bp; 1. Homologous recombination left arm; 2. EGFP expression cassette; 3. Homologous recombination right arm"
Fig. 3
The amplification results of the overlapping PCR A. Pre-fusion PCR agarose gel electrophoresis: M. DL 5000 bp DNA Marker; 1. Homologous recombination left arm+EGFP expression cassette; 2. EGFP expression cassette+Homologous recombination right arm; B. Fused PCR agarose gel electrophoresis: M. DL5000bp DNA Marker; 1. Homologous recombination left arm+EGFP expression cassette+Homologous recombination right arm; 2. EGFP expression cassette+Homologous recombination right arm+Homologous recombination left arm"
Fig. 4
The double digestion of the pUC19T vector and the results of PCR identification A. Kpn Ⅰ and BamH Ⅰ double digestion: M. DL5000 bp DNA Marker; 1. Target fragment; B. PCR agarose gel electrophoresis: M. DL5000 bp DNA Marker; 1-6. Positive bacteria solution"
Fig. 5
The rescue of the LSDV strain deleted ORF123 gene (10×)"
Fig. 6
The purification of the recombinant LSDV strain deleted ORF123 gene A, D. Recombinant virus white light (10×); B, E. Recombinant virus green fluorescence (10×); C, F. Recombinant virus white light+green light (10×); a, d. Recombinant virus white light (20×); b, e. Recombinant virus green fluorescence (20×); c, f. Recombinant virus white light+green light (20×) (Cytopathic effect was indicated by the arrow)"
Fig. 7
Identification of the recombinant strain LSDVΔORF123-EGFP by PCR A. The recombination strain LSDVΔORF123-EGFP was identified by primers F1/R1: M. DL5000bp DNA Marker; 1-3. The recombination strain LSDVΔORF123-EGFP was identified by primers F1/R1; 4. ddH2O; 5. Positive control; B. The recombination strain LSDVΔORF123-EGFP was identified by primers F2/R2: M. DL5000bp DNA Marker; 1-3. The recombination strain LSDVΔORF123-EGFP was identified by primers F2/R2; 4. ddH2O; 5. Positive control"
Fig. 8
Identification of the recombinant strain LSDVΔORF123-EGFP by Western blot"
Fig. 9
Growth curve of MDBK cells infected by LSDVΔORF123-EGFP strain and LSDV parental strains recombinant virus"
Fig. 10
Identification of the genetic stability for the recombinant strain LSDVΔORF123-EGFP (10×)"
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