Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Cultures and Conditions
2.2. Bovine Endothelial Cell Line
2.3. OMP Extraction
2.4. Binding Assays
2.5. Pull-Down Assays
2.6. Bioinformatics
2.7. Cloning, Expression, and Purification of Recombinant OMPs
2.8. Enzyme-Linked Immunosorbent Assays (ELISAs)
2.9. Adhesion Inhibition Assays
2.10. Statistical Analysis
3. Results
3.1. Identification and Characterization of OMPs from F. necrophorum subsp. necrophorum with the Ability to Bind a Host Endothelial Cell Line
3.2. Expression and Purification of Recombinant OMPs from F. necrophorum
3.3. Humoral Responses to Purified Recombinant Proteins in Immunized Rabbits and Immunoblotting
3.4. In Vitro Adhesion Inhibition Assay
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer | Tm |
---|---|---|
17 kDa F | 5’ TAGGATCCAGATAAAATTGCAGTGGTG 3’ | 56.9 °C |
17 kDa R | 5’ CGCTGCAGTTATTTTATTGTTTCCAT 3’ | 54.8 °C |
22 kDa F | 5’ TAGGATCCCGAAGAAGGAAATATAAAC 3’ | 54.3 °C |
22 kDa R | 5’ CGCTGCAGCTAGTTATATTTTGGAGC 3’ | 58.1 °C |
43 kDa F | 5’ TTGGGATCCTAAAGAAGTGATGCCTGCT 3’ | 61.5 °C |
43 kDa R | 5’ ACCTGCAGTTAGAAAGTAACTTTCATACC 3’ | 56.8 °C |
CSP F | 5’ GCCGGATCCAGAAGATCCGGTAATAAAAAGA 3’ | 61.2 °C |
CSP R | 5’ ACCTGCAGTTATTTGTTTATTAATTCTTC 3’ | 52.9 °C |
Accession | Description | # AAs | MW (kDa) |
---|---|---|---|
WP_035904032.1 | OmpH family protein | 159 | 17.5 |
WP_005961133.1 | OmpA family protein | 217 | 22.7 |
JQ740821.1 | 43 kDa outer membrane protein | 377 | 42.9 |
WP_035916891.1 | Cell surface protein | 638 | 66.3 |
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Bista, P.K.; Pillai, D.; Narayanan, S.K. Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum. Microorganisms 2023, 11, 2968. https://doi.org/10.3390/microorganisms11122968
Bista PK, Pillai D, Narayanan SK. Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum. Microorganisms. 2023; 11(12):2968. https://doi.org/10.3390/microorganisms11122968
Chicago/Turabian StyleBista, Prabha K., Deepti Pillai, and Sanjeev K. Narayanan. 2023. "Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum" Microorganisms 11, no. 12: 2968. https://doi.org/10.3390/microorganisms11122968