Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses, Cells and Plasmids
2.2. NP Gene Cloning and Recombinant Protein Expression
2.3. Mice Immunization
2.4. Cell Fusion and Identification of Monoclonal Antibodies
2.5. Enzyme-Linked Immunosorbent Assay (ELISA)
2.6. NP Gene Truncation Design and Expression
2.7. SDS-PAGE and Western Blotting
2.8. Indirect Immunofluorescence Assay (IFA)
2.9. Biological Information Analysis
3. Results
3.1. Cloning, Expression and Purification of Recombinant NP Protein
3.2. Screen of Monoclonal Antibody Specific to NP Proteins
3.3. Screening of B-Cell Determinants Specific to the Monoclonal Antibody
3.4. The Identified Epitope Is Highly Conserved in AIV Strains
3.5. Spatial Location Prediction of Epitope Binding
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene Name | Primer Name | Primer Sequence (5′–3′) | Primer Size | Tm(°C) | Product Size (bp) |
---|---|---|---|---|---|
NP | NP-F | CCGGAATTCTTTGATGAAAGGAGGAACAGATACC | 34 | 74.9 | 519 |
NP-R | CCCAAGCTTTCGCACTTGATCCATCATTGCTCTT | 34 | 75.1 | ||
NP-N-96 | NP-N-96-F | CCGGAATTCTTTGATGAAAGGAGGAACAG | 29 | 71.3 | 288 |
NP-N-96-R | ACGCGTCGACCAGAGAGCACATCCTGGGAT | 30 | 72 | ||
NP-C-103 | NP-C-103-F | CCGGAATTCTCCAACCTAAATGATGCCAC | 29 | 67.2 | 309 |
NP-C-103-R | ACGCGTCGACTCGCACTTGATCCATCATTG | 30 | 73.5 | ||
NP-C-54 | NP-C-54-F | CCGGAATTCCTGATGCAAGGATCAACTCTCCCGA | 34 | 72.3 | 162 |
NP-C-54-R | ACGCGTCGACATATGCAATCCTTGTTCTTCTTCCA | 35 | 74.5 | ||
NP-C-49 | NP-C-49-F | CCGGAATTCCGGATGATAAAGCGAGGGATCAACG | 34 | 72 | 147 |
NP-C-49-R | ACGCGTCGACTCGCACTTGATCCATCATTGCTCTT | 35 | 72.5 |
Monoclonal Antibody | Antibody of Cell Culture Supernatant Potency | Ascites Potency | Heavy Chain | Light Chain |
---|---|---|---|---|
4F5 | 1:6400 | 1:128,000 | IgG1 | κ |
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Huang, X.; Huang, J.; Yin, G.; Cai, Y.; Chen, M.; Hu, J.; Feng, X. Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus. Viruses 2022, 14, 1172. https://doi.org/10.3390/v14061172
Huang X, Huang J, Yin G, Cai Y, Chen M, Hu J, Feng X. Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus. Viruses. 2022; 14(6):1172. https://doi.org/10.3390/v14061172
Chicago/Turabian StyleHuang, Xiangyu, Jingwen Huang, Guihu Yin, Yiqin Cai, Mengli Chen, Jianing Hu, and Xiuli Feng. 2022. "Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus" Viruses 14, no. 6: 1172. https://doi.org/10.3390/v14061172
APA StyleHuang, X., Huang, J., Yin, G., Cai, Y., Chen, M., Hu, J., & Feng, X. (2022). Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus. Viruses, 14(6), 1172. https://doi.org/10.3390/v14061172