Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Virus
2.2. DNA Preparation, Restriction Digests, Southern Blotting
2.3. DNA Sequencing
2.4. Novel Synthetic ORFV Early Promoter eP1 and eP2
2.5. Construction of Transfer Plasmids
2.6. Nucleofection
2.7. Western Blotting
2.8. Flow Cytometry
2.9. Polymerase Chain Reaction (PCR)
2.10. Selection of ORFV Recombinants
3. Results
3.1. Detection and Mapping of Three Major Deletions in the D1701-V Genome.
3.2. ORFV Genes Affected by the 3 Deletions
3.3. Use of Deletion D for Foreign Gene Expression
3.4. Evaluation of Expression Strength Dictated by the used ORFV Early Promoters
4. Discussion
5. Conclusions and Future Perspectives
Patent
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Promoter eP1 | 5′-AAAAAAAAAATTGAAAAATTATTCTAAATATTGCACGG-3′ |
Promoter eP2 | 5′-AAAAATTGAAATTCTAACTTGTGTTCTTATAAATGAT-3′ |
Spacer SP | 5′-TTTTTATCTTGTTTTTATCCTGTCTTTTTATCAGTTTTTTA GCTAGTTAAAACATAAATAGTAAAGCTAAAAAGAGACTATATCGGCGGCTGGAGTCTTGCAACAACCAGC-3′ |
Ch-F | 5′-GCTTCAAGGTGCACATGGAGGGC-3′ |
Ch-R | 5′-GGTGTAGTCCTCGTTGTGGGAGG-3′ |
GFP-F | 5′-CCACAAGTTCAGCGTGAGCGGCGAG-3′ |
GFP-R | 5′-GCGCTTCTCGTTGGGGTCCTTGGAC-3′ |
Del2-F | 5′-GCAACAAGGTCTGCGTGCCTGCCGACC-3′ |
Del2-R2 | 5′-CGGCGATCTGGATGGTTGGAGCCATGCC-3′ |
pdV-F | 5′-GGTGACGGTGCTCAGCGTGGTGGCGGTTTC-3′ |
pdV-R | 5′-CTAGCGGCGTCTTCTGGGCGGCCTTGTGGT-3′ |
Open reading frame (ORF) | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
101 | 102 | 103 | 114 | 115 | 116 | 117 | 118 | |||||||||||||||||
ORFV strain | Length | Identity a) | Length | Identity | Length | Identity | Length | Identity | Length | Identity | Length | Identity | Length | Identity | Length | Identity | ||||||||
nt | aa | nt | nt | aa | nt | nt | aa | nt | nt | aa | nt | nt | aa | nt | nt | aa | nt | nt | aa | nt | nt | aa | nt | |
D1701-V | 3483 | 1161 | 100% | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | ― | 306 | 102 | 100% |
D1701-B | 3483 | 1161 | 100% | 1557 | 519 | 100% | 1548 | 516 | 100% | 1038 | 346 | 100% | 447 | 148 | 100% | 672 | 224 | 100% | 795 | 265 | 100% | 306 | 102 | 100% |
D1701-McG | 3483 | 1161 | 99% | 1557 | 519 | 100% | 1548 | 516 | 99% | 1038 | 346 | 99% | 447 | 148 | 100% | 672 | 224 | 99% | 912 | 304 | 99% | 306 | 102 | 100% |
NZ2 | 3483 | 1161 | 99% | 1560 | 520 | 95% | 1548 | 516 | 98% | 1038 | 346 | 96% | 435 | 145 | 88% | 693 | 231 | 81% | 795 | 265 | 96% | 357 | 119 | 97% |
OV-IA82 | 3483 | 1161 | 99% | 1556 | 518 | 85% | 1566 | 522 | 65% | 1038 | 346 | 96% | 429 | 143 | 89% | 702 | 234 | 81% | 795 | 265 | 96% | 663 | 221 | 97% |
B029 | 3483 | 1161 | 99% | 1560 | 520 | 95% | ― | ― | ― | 1038 | 346 | 97% | 435 | 145 | 89% | 669 | 223 | 83% | 795 | 265 | 96% | ― | ― | ― |
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Rziha, H.-J.; Büttner, M.; Müller, M.; Salomon, F.; Reguzova, A.; Laible, D.; Amann, R. Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression. Viruses 2019, 11, 127. https://doi.org/10.3390/v11020127
Rziha H-J, Büttner M, Müller M, Salomon F, Reguzova A, Laible D, Amann R. Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression. Viruses. 2019; 11(2):127. https://doi.org/10.3390/v11020127
Chicago/Turabian StyleRziha, Hanns-Joachim, Mathias Büttner, Melanie Müller, Ferdinand Salomon, Alena Reguzova, Dominic Laible, and Ralf Amann. 2019. "Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression" Viruses 11, no. 2: 127. https://doi.org/10.3390/v11020127
APA StyleRziha, H.-J., Büttner, M., Müller, M., Salomon, F., Reguzova, A., Laible, D., & Amann, R. (2019). Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression. Viruses, 11(2), 127. https://doi.org/10.3390/v11020127