Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Reagents
2.2. Preparation of Immune Serums
2.3. pB125R Prokaryotic Expression Vector Construction
2.4. pB125R Protein Expression and Purification
2.5. Production of mAbs against the ASFV pB125R Protein
2.6. Indirect ELISA
2.7. Western Blotting
2.8. Immunofluorescence Assay (IFA)
2.9. Antigenic Epitope Identification
2.10. Bioinformatic Analysis of the pB125R Protein
2.11. Immunogenicity of the Epitope of pB125R
2.12. Flow Cytometry
3. Results
3.1. The Expression and Purification of ASFV pB125R Recombinant Protein
3.2. Antibody Titers of the Serum from Mice Immunized with pB125R Protein
3.3. Preparation and Identification of mAbs against the pB125R Recombinant Protein
3.4. Mapping mAb 9G11epitopes
3.5. Bioinformatics Analysis of the Epitope in Different ASFV Strains
3.6. Verification of the Epitope Immunoactivity
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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Title | Primers | Sequences (5′–3′) |
---|---|---|
pB125R | F | ATCGGATCCGAATTCATGGCGGTTTATGCG |
R | GTGGTGGTGCTCGAGTCTAGACTCTAAAAATT |
Segment | Sequences (5′-3′) | Positions (Amino Acid) | |
---|---|---|---|
pB125R-1 | F | ATCGGATCCGAATTCATGGCGGTTTATGCG | 1-40AA |
R | GTGGTGGTGCTCGAGATACACCAAAAAGTT | ||
pB125R-2 | F | GTGGTGGTGCTCGAGATACACCAAAAAGTT | 31-70AA |
R | GTGGTGGTGCTCGAGCTCCTCGAGGCTTTT | ||
pB125R-3 | F | ATCGGATCCGAATTCTACTATGCCATCATA | 61-100AA |
R | GTGGTGGTGCTCGAGTTTGTTTATCTCAAT | ||
PB125R-4 | F | ATCGGATCCGAATTCAAAAAAATACAATCC | 91-125AA |
R | GTGGTGGTGCTCGAGTCTAGACTCTAAAAA | ||
pB125R-2-1 | F | AATTCAACTTTTTGGTGTATGAACTACCTGCCCCTTTTGACTTTTCCTCCC | 36-50AA |
R | TCGAGGGAGGAAAAGTCAAAAGGGGCAGGTAGTTCATACACCAAAAAGTTG | ||
pB125R-2-2 | F | AATTCGAACTACCTGCCCCTTTTGACTTTTCCTCCGGCGACCCTTTGGCCC | 41-55AA |
R | TCGAGGGCCAAAGGGTCGCCGGAGGAAAAGTCAAAAGGGGCAGGTAGTTCG | ||
pB125R-2-3 | F | AATTCTTTGACTTTTCCTCCGGCGACCCTTTGGCCAGTCAGCGCGACATAC | 46-60AA |
R | TCGAGTATGTCGCGCTGACTGGCCAAAGGGTCGCCGGAGGAAAAGTCAAAG | ||
pB125R-2-4 | F | AATTCGGCGACCCTTTGGCCAGTCAGCGCGACATATACTATGCCATCATAC | 51-65AA |
R | TCGAGTATGATGGCATAGTATATGTCGCGCTGACTGGCCAAAGGGTCGCCG | ||
pB125R-2-5 | F | AATTCGACCCTTTGGCCAGTCAGCGCGACATATACTATGCCATCC | 52-64AA |
R | TCGAGGATGGCATAGTATATGTCGCGCTGACTGGCCAAAGGGTCG | ||
pB125R-2-5-1 | F | AATTCCCTTTGGCCAGTCAGCGCGACATATACTATGCCATCC | 53-64AA |
R | TCGAGGATGGCATAGTATATGTCGCGCTGACTGGCCAAAGGG | ||
pB125R-2-5-2 | F | AATTCTTGGCCAGTCAGCGCGACATATACTATGCCATCC | 54-64AA |
R | TCGAGGATGGCATAGTATATGTCGCGCTGACTGGCCAAG | ||
pB125R-2-5-3 | F | AATTCGCCAGTCAGCGCGACATATACTATGCCATCC | 55-64AA |
R | TCGAGGATGGCATAGTATATGTCGCGCTGACTGGCG | ||
pB125R-2-5-4 | F | AATTCAGTCAGCGCGACATATACTATGCCATCC | 56-64AA |
R | TCGAGGATGGCATAGTATATGTCGCGCTGACTG | ||
pB125R-2-5-5 | F | AATTCCAGCGCGACATATACTATGCCATCC | 57-64AA |
R | TCGAGGATGGCATAGTATATGTCGCGCTGG | ||
pB125R-2-5-6 | F | AATTCCGCGACATATACTATGCCATCC | 58-64AA |
R | TCGAGGATGGCATAGTATATGTCGCGG | ||
pB125R-2-5-7 | F | AATTCGACATATACTATGCCATCC | 59-64AA |
R | TCGAGGATGGCATAGTATATGTCG | ||
pB125R-2-5-8 | F | AATTCATATACTATGCCATCC | 60-64AA |
R | TCGAGGATGGCATAGTATATG | ||
pB125R-2-5-9 | F | AATTCGACCCTTTGGCCAGTCAGCGCGACATATACTATGCCC | 52-63AA |
R | TCGAGGGCATAGTATATGTCGCGCTGACTGGCCAAAGGGTCG | ||
pB125R-2-5-10 | F | AATTCGACCCTTTGGCCAGTCAGCGCGACATATACTATC | 52-62AA |
R | TCGAGATAGTATATGTCGCGCTGACTGGCCAAAGGGTCG | ||
pB125R-2-5-11 | F | AATTCGACCCTTTGGCCAGTCAGCGCGACATATACC | 52-61AA |
R | TCGAGGTATATGTCGCGCTGACTGGCCAAAGGGTCG |
Title | Amino Acid Sequence | |
---|---|---|
pB125R-1 | MAVYAKDLDNNKELNQKLINDQLKIIDTLLLAEKKNFLVY | 1-40AA |
pB125R-2 | LAEKKNFLVYELPAPFDFSSGDPLASQRDIYYAIIKSLEE | 31-70AA |
pB125R-3 | YYAIIKSLEERGFTVKICMKGDRALLFITWKKIQSIEINK | 61-100AA |
PB125R-4 | KKIQSIEINKKEEYLRMHFIQDEEKAFYCKFLESR | 91-125AA |
pB125R-2-1 | NFLVYELPAPFDFSS | 36-50AA |
pB125R-2-2 | ELPAPFDFSSGDPLA | 41-55AA |
pB125R-2-3 | FDFSSGDPLASQRDI | 46-60AA |
pB125R-2-4 | GDPLASQRDIYYAII | 51-65AA |
pB125R-2-5 | DPLASQRDIYYAI | 52-64AA |
pB125R-2-5-1 | PLASQRDIYYAI | 53-64AA |
pB125R-2-5-2 | LASQRDIYYAI | 54-64AA |
pB125R-2-5-3 | ASQRDIYYAI | 55-64AA |
pB125R-2-5-4 | SQRDIYYAI | 56-64AA |
pB125R-2-5-5 | QRDIYYAI | 57-64AA |
pB125R-2-5-6 | RDIYYAI | 58-64AA |
pB125R-2-5-7 | DIYYAI | 59-64AA |
pB125R-2-5-8 | IYYAI | 60-64AA |
pB125R-2-5-9 | DPLASQRDIYYA | 52-63AA |
pB125R-2-5-10 | DPLASQRDIYY | 52-62AA |
pB125R-2-5-11 | DPLASQRDIY | 52-61AA |
Accession Number | Title | Country | Collection Date | Lengths of pB125R(AA) |
---|---|---|---|---|
MK128995.1 | ASFV China/2018/AnhuiXCGQ | China | 2021 | 125 |
FR682468.2 | ASFV Georgia 2007 | UK | 2020 | 125 |
LR536725.1 | ASFV Belgium 2018 | Germany | 2019 | 125 |
ON456300.2 | ASFV China/Yangzhou | China | 2023 | 125 |
LR899193.1 | ASFV Germany 2020 | Germany | 2020 | 125 |
MW049116.1 | ASFV Korea/2019 | Korea | 2022 | 125 |
OP856591.1 | China/LN/2018 | China | 2023 | 125 |
MT748042.2 | ASFV/Korea/2019 | Korea | 2023 | 125 |
MN715134.1 | ASFV_HU_2018 | Hungary | 2021 | 125 |
ON380539.1 | HB03A | China | 2022 | 125 |
MN393476.1 | ASFV Wuhan 2019 | China | 2020 | 125 |
OP605386.1 | 20355/RM/2022_Italy | Italy | 2023 | 125 |
MZ614662.1 | CADC_HN09 | China | 2021 | 125 |
MW396979.1 | ASFV/Timor-Leste/2019 | Australia | 2022 | 125 |
LS478113.1 | Estonia 2014 | Germany | 2018 | 125 |
ON409979.1 | TAN/17/Kibaha | Tanzania | 2023 | 125 |
LR813622.1 | Tanzania/Rukwa/2017 | Kenya | 2020 | 125 |
MT180393.1 | ASFV_NgheAn_2019 | S. Korea | 2021 | 125 |
MH681419.1 | ASFV/POL/2015/Podlaskie | Denmark | 2019 | 125 |
MW788407.1 | 31479_2005 | Italy | 2021 | 125 |
MZ202520.1 | K49 | Russia | 2021 | 125 |
ON075797.1 | Korea/YC1/2019 | Korea | 2022 | 125 |
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Zhao, Y.; Ren, H.; Lin, Z.; Shi, S.; Zhang, B.; Zhang, Y.; Han, S.; He, W.-R.; Wan, B.; Hu, M.; et al. Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody. Viruses 2024, 16, 1257. https://doi.org/10.3390/v16081257
Zhao Y, Ren H, Lin Z, Shi S, Zhang B, Zhang Y, Han S, He W-R, Wan B, Hu M, et al. Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody. Viruses. 2024; 16(8):1257. https://doi.org/10.3390/v16081257
Chicago/Turabian StyleZhao, Yanyan, Haojie Ren, Zhizhao Lin, Saiyan Shi, Biao Zhang, Yuhang Zhang, Shichong Han, Wen-Rui He, Bo Wan, Man Hu, and et al. 2024. "Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody" Viruses 16, no. 8: 1257. https://doi.org/10.3390/v16081257