A Novel Bacterium-Like Particle Vaccine Displaying the MERS-CoV Receptor-Binding Domain Induces Specific Mucosal and Systemic Immune Responses in Mice
Abstract
:1. Introduction
2. Materials and Methods
2.1. Construction and Expression of Recombinant Baculovirus
2.2. Immunofluorescence Assay (IFA) and Western Blotting Analyses of Recombinant Baculoviruses
2.3. Binding of the Fusion Proteins to Gem Particles
2.4. SDS-PAGE, Western Blotting and IFA Analysis of the Binding GEM Particles
2.5. Immunizations and Samples Collection
2.6. Pseudotyped Virus Neutralization Assay
2.7. Enzyme-Linked Immunosorbent Assay (ELISA) Measurement of RBD-Specific Antibodies
2.8. ELISpot IFN-γ and IL-4 Cytokine Assays
2.9. Splenocyte Proliferation Assay
2.10. Evaluation of B Cell and T Cell Activation by Flow Cytometry
2.11. ELISA Measurement of Cytokine Levels in Splenocyte Culture Supernatants
2.12. Data Analysis
2.13. Laboratory Facility and Ethics Statement
3. Results
3.1. Expression of Fusion Proteins
3.2. Location of Fusion Proteins on GEM Particles
3.3. Binding Activity of Fusion Proteins on GEM Particles
3.4. Virus Neutralizing Antibodies and ELISA Measurement of Subtype Antibodies in Serum
3.5. Mucosal IgA Levels in Lungs and Intestines
3.6. Splenocyte Proliferation by ex vivo Restimulation
3.7. Splenocyte Activation Assays
3.8. Cytokine Secretion by Restimulated Splenocytes
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Oligonucleotide Primers | Sequences |
---|---|
RBD-F1,3 | 5′-TGCTCTAGACATCACCATCACCATCACCAAGCCGAAGGAGTTGAA-3′ (XbaI) |
Linker-RBD-R2 | 5′-ACCAGAACCACCACCAGAACCACCCAACTTAGGGCAGACGCT-3′ |
RBD-R | 5′-GTTACCAGCTGAAGAAGCACCATCCAACTTAGGGCAGACGCT-3′ |
PA3-F | 5′-ACCAATAGCGTCTGCCCTAAGTTGGATGGTGCTTCTTCAGCTGG-3′ |
Linker-PA2-F2 | 5′-GGTGGTTCTGGTGGTGGTTCTGGTACTACCGTTAAGGTGAAGTC-3′ |
Linker-PA3-F2 | 5′-GGTGGTTCTGGTGGTGGTTCTGGTGATGGTGCTTCTTCAGCTGG-3′ |
PA3-R1 | 5′-CGGGGTACCTTACTTGATACGCAGGTATTGAC-3′ (KpnI) |
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Li, E.; Chi, H.; Huang, P.; Yan, F.; Zhang, Y.; Liu, C.; Wang, Z.; Li, G.; Zhang, S.; Mo, R.; et al. A Novel Bacterium-Like Particle Vaccine Displaying the MERS-CoV Receptor-Binding Domain Induces Specific Mucosal and Systemic Immune Responses in Mice. Viruses 2019, 11, 799. https://doi.org/10.3390/v11090799
Li E, Chi H, Huang P, Yan F, Zhang Y, Liu C, Wang Z, Li G, Zhang S, Mo R, et al. A Novel Bacterium-Like Particle Vaccine Displaying the MERS-CoV Receptor-Binding Domain Induces Specific Mucosal and Systemic Immune Responses in Mice. Viruses. 2019; 11(9):799. https://doi.org/10.3390/v11090799
Chicago/Turabian StyleLi, Entao, Hang Chi, Pei Huang, Feihu Yan, Ying Zhang, Chuanyu Liu, Zhenshan Wang, Guohua Li, Shengnan Zhang, Ruo Mo, and et al. 2019. "A Novel Bacterium-Like Particle Vaccine Displaying the MERS-CoV Receptor-Binding Domain Induces Specific Mucosal and Systemic Immune Responses in Mice" Viruses 11, no. 9: 799. https://doi.org/10.3390/v11090799