Title Page
Contents
Introduction 7
Materials and Methods 12
Bacteria, plasmids, and oligonucleotides 12
Construction of ΔmsbB mutant of S. enteritidis 13
Analysis of LPS 17
Analysis of lipid A by a thin-layer chromatography (TLC) 18
In vivo virulence assays 20
Preparation of OMVs 21
Electron microscopy 22
Immunization of mice and oral challenge with S. enteritidis 23
Measurement of serum IgG by ELISA 25
Serum bactericidal assay 26
Statistical analysis 27
Results 28
Confirmation of the ΔmsbB mutation in S. enteritidis 28
LPS alterations associated with the ΔmsbB mutation in S. enteritidis 30
Virulence assessment of the S. enteritidis ΔmsbB mutant 32
OMV production in the S. enteritidis ΔmsbB mutant 34
OMV vaccination induced Salmonella-specific immunity in mice leading to protection from oral challenge with wild type S. enteritidis 36
Discussion 42
References 47
Abstract 55
국문요약 58
ACKNOWLEDGEMENT 61
LIST OF Tables
Table 1. Serum bactericidal capacity 38
LIST OF Figures
Fig. 1. The route to human infection by S. enteritidis. A diversity of environments, living and inanimate, are colonized or infected. Eggs are said to be contaminated rather than infected, as they are not fertile and do not contain living cells... 9
Fig. 2. Protocol scheme for construction of the Salmonella mutant with a disrupted msbB gene 16
Fig. 3. Confirmation of the ΔmsbB mutant by genomic PCR. (A) The genomic PCR with P1 and P2 primers detected the cat gene (1,014 bp) only inserted in the ΔmsbB mutant (MT)... 29
Fig. 4. Analysis of LPS and the lipid A molecules. (A) Silver-stained LPS separated on 16% Tricine SDS-PAGE gel. (B) 32P-labeled lipid A molecules separated by a TLC as described previously (Zhou et al., 1999)... 31
Fig. 5. Survival rates of mice in lethality tests. (A) Survival rate of mice determined after i.p. injection with 108 CFU of the wild type (WT) (n=10) and the ΔmsbB mutant (MT) of the salmonellae (n=10)... 33
Fig. 6. TEM observations of the OMVs. The OMVs released from the wild type S. enteritidis (A) and the ΔmsbB mutant (B) colonies grown on LB agar plate... 35
Fig. 7. Measurements of anti-Salmonella antibodies raised by the OMV immunization. (A) Serum IgG titer was detected by ELISA with the plate coated with the Salmonella wild type. Sera from mice immunized (i.p.) with the wild type... 41