Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Viruses, and Vaccine
2.2. Construction of the Infectious Clones
2.3. Generation of the Recombinant Viruses
2.4. Immunofluorescence Assay and Antigen-Capture ELISA
2.5. Growth Curves of the Recombinant Viruses
2.6. Inoculation and Challenge Experiment in Rabbits
2.7. Blocking Enzyme-Linked Immunosorbent Assay
2.8. Immunoperoxidase Monolayer Assay (IPMA)
2.9. Virus Neutralization Assay
2.10. Statistical Analysis
3. Results
3.1. Generation of the Recombinant Viruses from the Cloned cDNAs
3.2. Stability of the Inserted Genes of the Recombinant Viruses in SK6 Cells
3.3. Expression of the Cap Protein of the Recombinant Viruses
3.4. Growth Curves of the Recombinant Viruses
3.5. The Phenotypes of the Recombinant Viruses in Rabbits
3.6. Immunogenicity of the Recombinant Viruses in Rabbits
4. Discussion
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Primers | Sequences (5′→3′) |
---|---|
pHCLV-2ACap-1F | CACCTCGAGATGCTACGTG |
pHCLV-2ACap-1R | CCCACTTGCGCCATCATCGGAGCAACTGGTAACCCACAATGG |
pHCLV-2ACap-2F | TCCGATGATGGCGCAAGTGGGACGTATCCAAGGAGGCGTTAC |
pHCLV-2ACap-2R | CCACATCGCCGGCCTGTTTCAGCAGCGAAAAGTTTGTGGCAGGGTTAAGTGGGGGTCTTTAAG |
pHCLV-2ACap-3F | GCTGAAACAGGCCGGCGATGTGGAGGAGAACCCAGGCCCATCCGATGATGGCGCAAGTGGGAG |
pHCLV-2ACap-3R | CTGATGCATGCACCTTGACAGTCGTGAATG |
pHCLV-uspCap-1F | CACCTCGAGATGCTACGTGGACGAGGG |
pHCLV-uspCap-1R | GGGCTGCAGCAGAAAGTATCACTGTAGACATTAAAATAGCTGAGATAATCTTTTTTTTCATCCCACTTGCGCCATCATC |
pHCLV-uspCap-2F | CTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCCCTGGAAATAAGCAGCACCTGCGATGCAGGCATCTTCAACACCC |
pHCLV-pspCap-2R | CTGATGCATGCACCTTGACAGTCGTG |
pHCLV-pspCap-1F | CACCTCGAGATGCTACGTGGACGAGGG |
pHCLV-pspCap-1R | GACCACCAGCAGCAGCAGCAGCCGTGATCCTTTCTGGGAGCTCCCCTTACTGTCCATCCCACTTGCGCCATCATCGGAG |
pHCLV-pspCap-2F | GGCTGCTGCTGCTGCTGGTGGTCAGCAACCTGCTGCTGCCTCAGGGGGTGGTCGGAGGCATCTTCAACACCCGCCTCTC |
pHCLV-pspCap-2R | CTGATGCATGCACCTTGACAGTCGTG |
Group | Prime Immunization | Dose (TCID50) | Number | Booster Immunization | Dose (TCID50) |
---|---|---|---|---|---|
A | rHCLV-2ACap | 104 | 6 | rHCLV-2ACap | 104 |
B | rHCLV-pspCap | 104 | 6 | rHCLV-pspCap | 104 |
C | rHCLV-uspCap | 104 | 6 | rHCLV-uspCap | 104 |
D | rHCLV | 104 | 6 | rHCLV | 104 |
E | C-strain | 104 | 6 | C-strain | 104 |
F | LG-vaccine | 1 mL | 3 | LG-vaccine | 1 mL |
G | DMEM | 1 mL | 6 | DMEM | 1 mL |
Group | Viruses | Dose (TCID50) | Fever Response | Viral Replication | Seroconversion of Antibodies Against CSFV E2 (PCV2 Cap) |
---|---|---|---|---|---|
A | rHCLV-2ACap | 104 | 4/6 | 3/3 | 3/3 (0/3) |
B | rHCLV-pspCap | 104 | 5/6 | 3/3 | 3/3 (1/3) |
C | rHCLV-uspCap | 104 | 4/6 | 3/3 | 3/3 (2/3) |
D | rHCLV | 104 | 5/6 | 3/3 | 3/3 (0/3) |
E | C-strain | 104 | 6/6 | 3/3 | 3/3 (0/3) |
F | LG-vaccine | 1 mL | n.d. | n.d. | n.d. (3/3) |
G | DMEM | 1 mL | 0/6 | 0/3 | 0/3 (0/3) |
Groups | Viruses | Days Post-Prime Immunization (Weeks Post-Booster Immunization) | |||||
---|---|---|---|---|---|---|---|
0 | 7 | 14 | 21 (0) | 28 (1) | 35 (2) | ||
A | rHCLV-2ACap | <10 | <10 | 75 ± 32 | 158 ± 65 | 710 ± 352 | >1028 |
B | rHCLV-pspCap | <10 | <10 | 89 ± 50 | 158 ± 95 | 670 ± 252 | >1028 |
C | rHCLV-uspCap | <10 | 20 | 89 ± 50 | 182 ± 56 | 630 ± 0 | >1028 |
E | C-strain | <10 | 21 ± 4 | 89 ± 50 | 188 ± 0 | 710 ± 352 | >1028 |
G | DMEM | <10 | <10 | <10 | <10 | <10 | <10 |
Groups | Viruses | Days Post-Prime Immunization (Weeks Post-Booster Immunization) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
0 | 7 | 14 | 21 (0) | 28 (1) | 35 (2) | 42 (3) | 49 (4) | 56 (5) | ||
A | rHCLV-2ACap | <10 | <10 | <10 | <10 | <10 | <10 | <10 | <10 | <10 |
B | rHCLV-pspCap | <10 | <10 | <10 | <10 | <10 | <10 | 80 ± 0 | 80 ± 0 | 160 ± 0 |
C | rHCLV-uspCap | <10 | <10 | <10 | <10 | <10 | 20 ± 0 | 80 ± 400 | 160 ± 0 | 160 ± 0 |
F | LG-vaccine | <10 | <10 | <10 | <10 | <10 | 40 ± 88 | 320 ± 0 | 640 ± 0 | 640 ± 0 |
G | DMEM | <10 | <10 | <10 | <10 | <10 | <10 | <10 | <10 | <10 |
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Zhang, L.; Li, Y.; Xie, L.; Wang, X.; Gao, X.; Sun, Y.; Qiu, H.-J. Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector. Viruses 2017, 9, 298. https://doi.org/10.3390/v9100298
Zhang L, Li Y, Xie L, Wang X, Gao X, Sun Y, Qiu H-J. Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector. Viruses. 2017; 9(10):298. https://doi.org/10.3390/v9100298
Chicago/Turabian StyleZhang, Lingkai, Yongfeng Li, Libao Xie, Xiao Wang, Xulei Gao, Yuan Sun, and Hua-Ji Qiu. 2017. "Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector" Viruses 9, no. 10: 298. https://doi.org/10.3390/v9100298
APA StyleZhang, L., Li, Y., Xie, L., Wang, X., Gao, X., Sun, Y., & Qiu, H. -J. (2017). Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector. Viruses, 9(10), 298. https://doi.org/10.3390/v9100298