Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses and Cells
2.2. Expression and Purification of Recombinant M Protein
2.3. Preparation of Monoclonal Antibodies Against the M Protein
2.4. Screening of Anti-RABV M Antibodies Using Indirect ELISA
2.5. SDS-PAGE and Western Blotting
2.6. Indirect Immunofluorescence Assay
2.7. Identification of M Protein Epitopes
2.8. Alignment of Epitope Sequences of the M Protein
2.9. Application of mAbs in the Detection of RABV by Immunohistochemistry
3. Results
3.1. Expression and Purification of the M Protein
3.2. Generation and Characterization of mAbs Against the M Protein
3.3. Mapping of the Epitopes of M Protein
3.4. Alignment of M Protein Epitope Sequences
3.5. Reactivity of mAbs with RABV in the Brain Tissue of Infected Mice Using Immunohistochemistry (IHC)
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Fragment | Primer Sequence (5′–3′) |
---|---|
1–203 aa | GGGGTACCATGAACGTTCTACGCAAGAT (HindIII) |
CCCAAGCTTTTATTCTAGAAGCAGAGAAGAG (KpnI) | |
1–107 aa | GGGGTACCATGAACGTTCTACGCAAGAT (HindIII) |
CCCAAGCTTAGGTACTGGAGCTCCTGATAAA (KpnI) | |
101–203 aa | GGGGTACCTTATCAGGAGCTCCAGTACCT (HindIII) |
CCCAAGCTTTTATTCTAGAAGCAGAGAAGAG (KpnI) | |
1–32 aa | GGGGTACCATGAACGTTCTACGCAAGATA (HindIII) |
CCCAAGCTTCCACAGGTCATCGTCATACGG (KpnI) | |
26–36 aa | GGGGTACCTATGACGATGACCTGTGGCTTCCACCTCCT (HindIII) |
CCCAAGCTTAGGAGGTGGAAGCCACAGGTCATCGTCATACGG (KpnI) | |
33–203 aa | GGGGTACCTGACCTGTGGCTTCCACCTC (HindIII) |
CCCAAGCTTTTATTCTAGAAGCAGAGAAGAG (KpnI) | |
1–17 aa | GGGGTACCATGAACGTTCTACGCAAGAT (HindIII) |
TAGGGATGAGGACACTCAAAAGCTTGGG (KpnI) | |
17–26 aa | GGGGTACCCAAAAGCCCTCTCCTGTGT (HindIII) |
CCCAAGCTTCCACAGGTCATCGTCATACGG (KpnI) |
Fragment | Primer Sequence (5′–3′) |
---|---|
17–25 aa | CCAAAAGCCCTCTCCTGTGTCAGCCCCTA |
CATGGGTTTTCGGGAGAGGACACAGTCGGGGATTCGA | |
24–32 aa | CGCCCCTCCGTATGACGATGACCTGTGGA |
CATGGCGGGGAGGCATACTGCTACTGGACACCTTCGA | |
24–31 aa | GATCCGCCCCTCCGTATGACGATGACCTGC |
TCGAGCAGGTCATCGTCATACGGAGGGGCG | |
24–30 aa | GATCCGCCCCTCCGTATGACGATGACC |
TCGAGGTCATCGTCATACGGAGGGGCG | |
24–29 aa | GATCCGCCCCTCCGTATGACGATC |
TCGAGATCGTCATACGGAGGGGCG | |
24–28 aa | GATCCGCCCCTCCGTATGACC |
TCGAGGTCATACGGAGGGGCG | |
25–32 aa | GATCCCCTCCGTATGACGATGACCTGTGGC |
TCGAGCCACAGGTCATCGTCATACGGAGGG | |
26–32 aa | GATCCCCGTATGACGATGACCTGTGGC |
TCGAGCCACAGGTCATCGTCATACGGG | |
27–32 aa | GATCCTATGACGATGACCTGTGGC |
TCGAGCCACAGGTCATCGTCATAG | |
28–32 aa | GATCCGACGATGACCTGTGGC |
25–30 aa | TCGAGCCACAGGTCATCGTCG GATCCCCTCCGTATGACGATGACC TCGAGGTCATCGTCATACGGAGGG |
25–30 aa (pCMV-Flag) | AGCTTCCTCCGTATGACGATGACGGTAC AGTCATCGTCATACGGAGGC |
Fragment | Primer Sequence (5′–3′) |
---|---|
P26L mutation | GATCCCCTCTGTATGACGATGACC |
TCGAGGTCATCGTCATACAGAGGG | |
Y27D mutation | GATCCCCTCCGGATGACGATGACC |
TCGAGGTCATCGTCATCCGGAGGG | |
D28G mutation | GATCCCCTCCGTATGGCGATGACC |
TCGAGGTCATCGCCATACGGAGGG |
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Liu, J.; Zhao, W.; He, W.; Wang, N.; Su, J.; Ji, S.; Chen, J.; Wang, D.; Zhou, J.; Su, S. Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus. Viruses 2019, 11, 375. https://doi.org/10.3390/v11040375
Liu J, Zhao W, He W, Wang N, Su J, Ji S, Chen J, Wang D, Zhou J, Su S. Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus. Viruses. 2019; 11(4):375. https://doi.org/10.3390/v11040375
Chicago/Turabian StyleLiu, Jie, Wen Zhao, Wanting He, Ningning Wang, Jingyin Su, Senlin Ji, Jian Chen, Dong Wang, Jiyong Zhou, and Shuo Su. 2019. "Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus" Viruses 11, no. 4: 375. https://doi.org/10.3390/v11040375