Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Cell Lines and Virus Strains
2.3. Animal Immunization and E2 Antibody Testing
2.4. Cell Staining and Porcine Single B Cell Sorting
2.5. Porcine IgG Gene-Specific Single B Cell RT-PCR
2.6. Cloning and Expression of Porcine Monoclonal Antibodies in Mammalian Expression System
2.7. Western Blotting and Indirect Immunofluorescence (IFA) Testing of Porcine Monoclonal Antibodies
3. Results
3.1. Isolation of CSFV C-Strain E2 Protein-Induced Single Porcine B Cells
3.2. Amplification of IgG Heavy and Light Chains from Porcine Single B Cell
3.3. Genetic Characterizon of the Paired Chains Recovered from CE2+IgG+ Porcine Single B Cells
3.4. The Porcine mAbs Can Specifically Bind to CSFV CE2 Protein
3.5. The Porcine mAbs Exhibited High Potency in Neutralizing CSFVs
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence (5′-3′) | PCR Product |
---|---|---|
D-WHF1 | CGGAATTCGCCATGGAGTTTCGGCTGAACTG | whole coding region of porcine IgH chain |
D-WHF2 | CGGAATTCGCCATGGGATTTCGGCTGAACTG | |
D-WHR1 | GCTCTAGATCATTTACCCGGAGTCTTGGAGATAGAC | |
D-WHR2 | GCTCTAGATCATTTACCCKGAGTCTKGRAGAYGGAC | |
D-WκF1 | CGGAATTCACCATGAGGTTCCCTGCTCAGCTCCTG | whole coding region of porcine Igκ chain |
D-WκF2 | CGGAATTCACCATGAGGGCCCCCATGCACCTCCTTG | |
D-WκF3 | CGGAATTCACCATGAGGGTCCCCGCTCAGCTCCTG | |
D-WκR1 | GCTCTAGACTAAGCCTCACACTCGTTCCTGYTGAAGCT | |
D-WκR2 | GCTCTAGACTAACACTCTCCTCTGTTGAAGCTCTTGG | |
D-WλF1 | CGGAATTCACCATGGCCTGGAYCCCTCTCCT | whole coding region of porcine Igλ chain |
D-WλF2 | CGGAATTCACCATGGCCTGGACGGTGCTTCTG | |
D-WλF3 | CGGAATTCACCATGAGGCCCAGGTCAGGCCAG | |
D-WλR1 | GCTCTAGACTAGGCGCACTCGGAGGGCRT |
Subclasses | IgG3 | H9 | H11 | IgG4 | IgG2b | IgG2c | IgG2a | IgG1 | H1 | IgG5a | IgG5b | IgG5c |
---|---|---|---|---|---|---|---|---|---|---|---|---|
IgG3 | ID | 0.80 | 0.80 | 0.81 | 0.83 | 0.83 | 0.82 | 0.82 | 0.79 | 0.75 | 0.75 | 0.75 |
H9 | 0.80 | ID | 1.00 | 0.95 | 0.88 | 0.90 | 0.89 | 0.87 | 0.85 | 0.82 | 0.81 | 0.81 |
H11 | 0.80 | 1.00 | ID | 0.95 | 0.88 | 0.89 | 0.89 | 0.87 | 0.85 | 0.82 | 0.80 | 0.81 |
IgG4 | 0.81 | 0.95 | 0.95 | ID | 0.91 | 0.94 | 0.91 | 0.88 | 0.84 | 0.80 | 0.79 | 0.81 |
IgG2b | 0.83 | 0.88 | 0.88 | 0.91 | ID | 0.97 | 0.96 | 0.92 | 0.86 | 0.84 | 0.80 | 0.81 |
IgG2c | 0.83 | 0.90 | 0.89 | 0.94 | 0.97 | ID | 0.94 | 0.90 | 0.86 | 0.82 | 0.78 | 0.81 |
IgG2a | 0.82 | 0.89 | 0.89 | 0.91 | 0.96 | 0.94 | ID | 0.92 | 0.86 | 0.85 | 0.81 | 0.81 |
IgG1 | 0.82 | 0.87 | 0.87 | 0.88 | 0.92 | 0.90 | 0.92 | ID | 0.92 | 0.87 | 0.83 | 0.84 |
H1 | 0.79 | 0.85 | 0.85 | 0.84 | 0.86 | 0.86 | 0.86 | 0.92 | ID | 0.82 | 0.80 | 0.80 |
IgG5a | 0.75 | 0.82 | 0.82 | 0.80 | 0.84 | 0.82 | 0.85 | 0.87 | 0.82 | ID | 0.90 | 0.88 |
IgG5b | 0.75 | 0.81 | 0.80 | 0.79 | 0.80 | 0.78 | 0.81 | 0.83 | 0.80 | 0.90 | ID | 0.89 |
IgG5C | 0.75 | 0.81 | 0.81 | 0.81 | 0.81 | 0.81 | 0.81 | 0.84 | 0.80 | 0.88 | 0.89 | ID |
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Wang, L.; Madera, R.; Li, Y.; Gladue, D.P.; Borca, M.V.; McIntosh, M.T.; Shi, J. Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells. Viruses 2023, 15, 863. https://doi.org/10.3390/v15040863
Wang L, Madera R, Li Y, Gladue DP, Borca MV, McIntosh MT, Shi J. Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells. Viruses. 2023; 15(4):863. https://doi.org/10.3390/v15040863
Chicago/Turabian StyleWang, Lihua, Rachel Madera, Yuzhen Li, Douglas P. Gladue, Manuel V. Borca, Michael T. McIntosh, and Jishu Shi. 2023. "Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells" Viruses 15, no. 4: 863. https://doi.org/10.3390/v15040863
APA StyleWang, L., Madera, R., Li, Y., Gladue, D. P., Borca, M. V., McIntosh, M. T., & Shi, J. (2023). Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells. Viruses, 15(4), 863. https://doi.org/10.3390/v15040863