Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity
Abstract
:1. Introduction
2. Results
2.1. Porcine RLR Protein Purification and Development of RLR-Specific Hybridoma
2.2. Characterization of mAb Reactivity with Porcine RLRs by Western Blotting
2.3. Immunofluorescence Using mAbs Revealed the Distinct Cytoplasmic Localizations of Porcine RLRs
2.4. Determination of the Recognition Regions on Porcine RLRs and Cross-Reactivity with Human RLRs by mAbs
2.5. Detection of RLR Expressions in Different Tissues from PRRSV-Infected Pigs Using mAbs
3. Discussion
4. Materials and Methods
4.1. Cells and Reagents
4.2. Molecular Cloning, RLR Protein Expressions and Purifications
4.3. Immunization, Cell Fusion and Screening of RLR Protein-Specific Hybridomas
4.4. ELISA and Western Blotting
4.5. Immunofluorescence Microscopy
4.6. Quantitative RT-PCR
4.7. Immunohistochemistry (IHC)
4.8. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer Names | Primer Sequences | Length |
---|---|---|
RIG-I-Δ2CARDs | F: catcattttggcaaaGAATTCatggaggatgacgaaatgaaaacttgt R: tatgggtagctggtGATATCctcaaggttgcccattc | 2232 bp |
MDA5-Δ2CARDs | F: cattttggcaaaGAATTCatgaccgattgctgtgaaagca R: tatgggtagctggtGATATCgtcctcatcactagacaaac | 2472 bp |
Genes Amplified | Primer Names | Primer Sequences (5′→3′) | Length |
---|---|---|---|
pRIG-I | pRIG-I-F | cccagtgtatgagcagcaga | 234 bp |
pRIG-I-R | ctggtgttgtggcattcatc | ||
pMDA5 | pMDA5-F | ctcaaagagcatcccctgag | 246 bp |
pMDA5-R | gttcgaactctttgcggaag | ||
pLGP2 | pLGP2-F | gttcgaactctttgcggaag | 150 bp |
pLGP2-R | gacccttgaactgcttctgc | ||
pβ-actin | pβ-actin-F | atgaagatcaagatcatcgcg | 116 bp |
pβ-actin-R | tcgtactcctgcttgctgatc |
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Shao, Q.; Li, S.; Cao, Q.; Gu, H.; Zhang, J.; Zhang, Y.; Zhang, K.; Zheng, W.; Chen, N.; Shang, S.; et al. Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity. Int. J. Mol. Sci. 2023, 24, 4118. https://doi.org/10.3390/ijms24044118
Shao Q, Li S, Cao Q, Gu H, Zhang J, Zhang Y, Zhang K, Zheng W, Chen N, Shang S, et al. Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity. International Journal of Molecular Sciences. 2023; 24(4):4118. https://doi.org/10.3390/ijms24044118
Chicago/Turabian StyleShao, Qi, Shuangjie Li, Qi Cao, Haotian Gu, Jiajia Zhang, Youwen Zhang, Kaili Zhang, Wanglong Zheng, Nanhua Chen, Shaobin Shang, and et al. 2023. "Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity" International Journal of Molecular Sciences 24, no. 4: 4118. https://doi.org/10.3390/ijms24044118