Title Page
Contents
Abstract 9
초록 11
Ⅰ. Introduction 14
Ⅱ. Materials & Methods 20
1. Bacterial and phage strains 20
2. Delivery of immunostimulatory CpG-ODNs via genetically engineered phage 21
2.1. Construction of immunostimulatory CpG motif harboring phagemid 21
2.2. Preparation of electro-competent TG1 cell 22
2.3. Electroporation of the phagemid DNA 23
2.4. Production of phagemid particles harboring stimulatory CpG ODN within its genome 24
2.5. Phage administration 26
2.6. Chemiluminescence (CL) assay 27
3. Delivery of heterologous antigen using phage display technology 28
3.1. Construction of SpyTag displaying phagemid on P8 28
3.2. Preparation of the SpyTag displaying phage 30
3.3. Vector construction for expressing SpyCatcher fused protein 32
3.4. Recombinant protein expression and purification 34
3.5. SDS-PAGE and Western blotting 35
3.6. Conjugation reaction between Spytag displaying phage and Spycatcher fused protein 36
3.7. Fish immunization 37
3.8. ELISA 38
3.9. Statistical Analysis 39
Ⅲ. Results 40
1. Delivery of immunostimulatory CpG-ODNs via genetically engineered phage 40
1.1. Chemiluminescence (CL) assay 40
2. Delivery of heterologous antigen using phage display technology 42
2.1. Expression and purification of GFP-SpyCatcher (GFP-Sc) 42
2.2. Conjugation reaction between p8-st and GFP-Sc 43
2.3. Specific serum antibody titer against GFP 44
2.4. Expression and purification of FlaB-SpyCatcher (FlaB_Sc) 45
2.5. Conjugation reaction between p8-st and FlaB-Sc 46
Ⅳ. Discussion 47
Ⅴ. Reference 52
Table 1. Primers used for pComb8-SpyTag phagemid construction 29
Table 2. Primers used for pFlaB-SpyCatcher protein expression vector construction 33
Figure 1. Effect of phage harboring immunostimulatory CpG-ODNs on the respiratory burst activity of olive flounder head kidney phagocytes... 41
Figure 2. SDS-PAGE (A) and Western blot analysis (B) of the purified GFP-SpyCatcher (GFP-Sc). 42
Figure 3. SDS-PAGE (A) and Western blot analysis of conjugation reaction between the p8-st and GFP-Sc. 43
Figure 4. Juvenile rainbow trout were immunized with GFP_Sc (GFP) or phage-displaying GFP (phage-GFP). 44
Figure 5. SDS-PAGE (A) and Western blot analysis (B) of the FlaB-SpyCatcher (FlaB-Sc). 45
Figure 6. SDS-PAGE (A) and Western blot analysis (B) of the p8-st and FlaB-Sc conjugate. 46