Title Page
Contents
I. Introduction 12
II. METERIALS AND METHODS 16
1. Cell lines 16
2. Virus acquisition and amplification 16
3. Plaque formation assay 17
4. Peptides 18
5. Antibodies 19
6. Co-immunoprecipitation 20
7. Analysis of MERS-CoV S protein binding proteins 21
8. Immunoprecipitation and Western blotting 22
9. Confirmation of interaction between Spike CD and N protein of MERS-CoV 23
10. Confirmation of interaction between Spike CD and N protein of SARS-CoV-2 24
11. Cell penetration assay 25
12. Cell viability assay 26
13. Analysis of S protein and N protein expression of MERS-CoV and SARS-CoV-2 with confocal microscopy 27
14. Inhibition effects of Spike CD-MERS-CoV peptides on MERS-CoV replication 28
15. Inhibition effects of Spike CD-SARS-CoV-2 peptides on SARS-CoV-2 replication 28
16. Quantitative RT-PCR 29
17. Animals 30
18. Virus challenge experiments 31
19. Hematoxylin and eosin staining 32
20. Immunohistochemistry 33
21. Expression of recombinant coronaviruses N proteins 34
22. Expression of human Fc domain-Spike CD of coronaviruses fusion proteins 36
23. Mice immunization 37
24. Production of SARS-CoV-2 N protein targeting mouse monoclonal antibody 37
25. Antigen-specific ELISA 38
26. Binding affinity ELISA 39
27. Pull-down assay 40
28. Bait and prey system for evaluation of the interaction between Spike CD and N protein of SARS-CoV-2 40
29. Detection of SARS-CoV-2 N protein using the interaction of Spike CD-N protein 42
30. Statistical analysis 43
III. RESULTS 44
1. Interaction between the S protein and N protein of MERS-CoV 44
2. Interaction of Spike C-terminal domain and N protein of MERS-CoV 45
3. Cell penetration and cytotoxicity analysis of Spike CD peptides of MERS-CoV and SARS-CoV-2 46
4. Inhibition of MERS-CoV replication by cell-penetrating R-Spike CD-MERS-CoV peptide 47
5. Interaction between the S protein and N protein of SARS-CoV-2 48
6. Inhibition of SARS-CoV-2 replication by cell-penetrating R-Spike CD-SARS-CoV-2 peptide 49
7. Effects of R-Spike CD-SARS-CoV-2 peptide on N protein expression of SARS-CoV-2 variants 51
8. Effects of R-Spike CD-SARS-CoV-2 peptide on SARS-CoV-2-infected K18-hACE2 transgenic mouse 52
9. Optimization of Spike CD peptide and inhibition of SARS-CoV-2 replication in vitro 53
10. Efficacy of R-Spike CD (D) against SARS-CoV-2 infection in K18-hACE2 mouse model 55
11. Generation of mouse monoclonal antibody against SARS-CoV-2 N protein 57
12. Specificity of the anti-SARS-CoV-2 N protein monoclonal antibody 58
13. Detection of N proteins in SARS-CoV-2 variants with anti-SARS-CoV-2 N protein monoclonal antibody 59
14. Evaluation of interaction between recombinant SARS-CoV-2 Spike CD fusion protein and recombinant SARS-CoV-2 N protein 61
15. Detection of SARS-CoV-2 N protein using the interaction of Spike CD-fusion protein and N protein of SARS-CoV-2 62
IV. DISCUSSION 64
V. REFERENCES 70
VI. Abstract Korean 119
VII. Abstract English 121
Figure 1. Interaction of S protein and N protein of MERS-CoV in MERS-CoV-infected cells. 80
Figure 2. Analysis of protein interacting with MERS-CoV S protein. 81
Figure 3. Time-dependent detection of MERS-CoV structural proteins on MERS-CoV-infected Vero cell lysates. 82
Figure 4. MERS-CoV N protein interacts with S and M proteins. 83
Figure 5. Analysis of S protein and N protein interaction of MERS-CoV. 84
Figure 6. Internalization and cytotoxicity of R-Spike-CD peptides in Vero cells and Calu-3 cells. 86
Figure 7. Inhibitory effects of cell-penetrating R-Spike-CD-MERS-CoV on MERS-CoV replication. 87
Figure 8. Interaction of SARS-CoV-2 S protein and N protein. 89
Figure 9. Effects of R-Spike-CD-SARS-CoV-2 peptide on the interaction of SARS-CoV-2 S protein and N protein. 90
Figure 10. Effects of R-Spike CD-SARS-CoV-2 peptide on SARS-CoV-2 replication. 91
Figure 11. Effects of R-Spike CD-SARS-CoV-2 peptide on SARS-CoV-2 S clade in Vero cells and Calu-3 cells. 93
Figure 12. Effects of R-Spike CD-SARS-CoV-2 on delta and iota variants of SARS-CoV-2. 95
Figure 13. Survival rate of SARS-CoV-2 S clade and delta variant-infected-K18-hACE2 transgenic mouse. 96
Figure 14. Effects of R-Spike CD-SARS-CoV-2 peptide on SARS-CoV-2 S clade-infected K18-hACE2 transgenic mouse. 97
Figure 15. Effects of R-Spike CD-SARS-CoV-2 peptide on SARS-CoV-2 delta variant-infected K18-hACE2 transgenic mouse. 98
Figure 16. Synthesis of Spike-CD peptide of SARS-CoV-2. 99
Figure 17. Internalization of R-Spike CD (D) in Vero cells and Calu-3 cells. 100
Figure 18. Inhibition of SARS-CoV-2 replication by R-Spike CD (D) peptide in Vero cells. 101
Figure 19. Effects of R-Spike CD (D) on body weight and survival in R-Spike CD (D) peptide-treated K18-hACE2 transgenic mouse against SARS-CoV-2 infection. 103
Figure 20. Effects of R-Spike CD (D) peptide on SARS-CoV-2-infected K18-hACE2 transgenic mouse. 104
Figure 21. Generation of monoclonal antibody against SARS-CoV-2 N protein. 106
Figure 22. Specificity of the purified anti-SARS-CoV-2 N protein monoclonal antibody. 109
Figure 23. Detection of N proteins in SARS-CoV-2 variants-infected cells with anti-SARS-CoV-2 N protein-specific monoclonal antibody. 111
Figure 24. Detection of anti-SARS-CoV-2 N protein in variants with anti-SARS-CoV-2 N protein-specific monoclonal antibody. 113
Figure 25. Interaction between Spike CD-fusion protein and N proteins of S clade, alpha variant, and beta variant of SARS-CoV-2. 115
Figure 26. Interaction analysis of SARS-CoV-2 Spike CD-Fc and SARS-CoV-2 N-Bio-His6 recombinant protein.[이미지참조] 116
Figure 27. Detection of SARS-CoV-2 N protein in cell culture supernatants using the bait and prey system. 118