Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins
Abstract
:1. Introduction
2. Results
2.1. TNP MAb 20C4C8 Recognizes the TSWV Serogroup of Euro-America Type Orthotospoviruses, but Not Asia Type Orthotospoviruses
2.2. Identification of the Common Epitope Recognized by the TNP MAb 20C4C8
2.3. The Amino Acid Sequence of NP200-229 Containing the Core Sequence KGKEYA Recognized by TNP MAb
2.4. The Feasibility of the NP Sequence for Tagging Different Recombinant Proteins in the Bacterial Expression System
2.5. Co-Immunoprecipitation of NP-Tagged or NSs-Tagged ZYMV CP In Vitro
3. Discussion
4. Materials and Methods
4.1. Virus Sources
4.2. Purification of Bacteria-Expressed TSWV NP
4.3. Production of Mouse Antisera and Monoclonal Antibodies
4.4. Reactions of the Selected TSWV-NP MAb 20C4C8 with Different Orthotospoviruses
4.5. Search for the Highly Conserved Domains of NPs by Sequence Alignment
4.6. Epitope Mapping Using Full Length or Truncated TSWV NP Fused with NSs-Tagged GFP
4.7. Identification of the Minimal Length of the Epitope
4.8. Evaluation of the NP Sequence for Tagging GFP, ZYMV CP, and Mite Chimeric Allergen Dp 25 in the Bacteria-Expressed System
4.9. Protein Expression and Detection by Western Blotting
4.10. Co-Immunoprecipitation of NP-Tagged Proteins In Vitro
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer a | Sequence (5′-3′) | Position of a.a. b | Rz c |
---|---|---|---|
TSWV NP expression in bacteria | |||
P-TNP-BamHI | AAGGGATCCATGTCTAAGGTTAAGCTCA | 1 | BamHI |
M-TNP-XhoI | CACTCGAGAGCAAGTTCTGTGAGTT | 258 | XhoI |
Epitope mapping of TSWV NP | |||
P-T7-pro. | TAATACGACTCACTATAGG | V | NcoI |
P-TNP-199 | AAGGGATCCATTAAGCAAAGTGATTTTACT | 67 | BamHI |
P-TNP-397 | AAGGGATCCCCTCTTGATGATGCAAAGT | 133 | BamHI |
P-TNP-598 | AAGGGATCCAAAGCATTTGAAATGAATG | 200 | BamHI |
P-TNP-634 | AGGGATCCGGAAAAGAGTATGCTGCTAT | 212 | BamHI |
P-TNP-637 | AGGGATCCAAAGAGTATGCTGCTATACT | 211 | BamHI |
P-TNP-652 | GGGATCCATACTTAGCTCCAGCAATCC | 218 | BamHI |
M-TNP-KpnI | CAGGTACCAGCAAGTTCTGTGAGTT | 258 | KpnI |
M-TNP-732 | CAGGTACCTTCATAGAACTTGTTAAGAGTTTC | 244 | KpnI |
M-TNP-687 | CAGGTACCACTTCCTTTAGCATTAGGATTG | 229 | KpnI |
M-TNP-642 | CAGGTACCCTCTTTTCCTTTCTTCACCTG | 214 | KpnI |
M-TNP-654 | CAGGTACCTATAGCAGCATACTCTTTTCCT | 218 | KpnI |
M-TNP-660 | CAGGTACCGCTAAGTATAGCAGCATACTC | 220 | KpnI |
M-TNP-645 | CAGGTACCATACTCTTTTCCTTTCTTCACC | 215 | KpnI |
M-pET-PsiI | AAAATCCCTTATAAATCAAAAGAAT | V | PsiI |
np sequence taggingd | |||
P-tnp-KpnI | GATCCGGTACCAAAGGAAAAGAGTATGCTTGACTCGAGCACCACCACCA | 211-216 | KpnI |
M-pET-PsiI | AAAATCCCTTATAAATCAAAAGAAT | V | PsiI |
P-pET-MluI | CAGCCCACTGACGCGTTGCGCGA | V | MluI |
M-tnp-NcoI | ACGCATGCCATGGCATACTCTTTTCCTTTCATGGTATATCTCCTTCTTA | 211-216 | NcoI |
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Cheng, H.-W.; Tsai, W.-T.; Hsieh, Y.-Y.; Chen, K.-C.; Yeh, S.-D. Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins. Int. J. Mol. Sci. 2021, 22, 8583. https://doi.org/10.3390/ijms22168583
Cheng H-W, Tsai W-T, Hsieh Y-Y, Chen K-C, Yeh S-D. Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins. International Journal of Molecular Sciences. 2021; 22(16):8583. https://doi.org/10.3390/ijms22168583
Chicago/Turabian StyleCheng, Hao-Wen, Wei-Ting Tsai, Yi-Ying Hsieh, Kuan-Chun Chen, and Shyi-Dong Yeh. 2021. "Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins" International Journal of Molecular Sciences 22, no. 16: 8583. https://doi.org/10.3390/ijms22168583