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병원성 파울러자유아메바 감염에 대한 rNfa1 단백질로 면역시킨 마우스의 방어 면역

Protective Immunity in Mice Immunized with the Nfa1 Protein for Pathogenic Naegleria fowleri Infection

초록/요약

Pathogenic Naegleria fowleri causes a fatal disease, called as primary amoebic menigoencephalitis, in humans and experimental animals. In the present study, to examine the protective immunity of the Nfa1 protein for N. fowleri infection in a mouse model, BALB/c mice were immunized with the Nfa1 protein either by intraperitoneal or intranasal route, and then infected intranasally with N. fowleri trophozoites. In regardless of the immunization routes, Nfa1-immunized mice showed the prolonged mean time to death. The specific immunoglobulin G (IgG), IgG subclass, IgA and IgE of mice induced against Nfa1 protein immunization was measured by ELISA. The levels of IgG and IgA were significantly increased in sera obtained from mice immunized with rNfa1 protein. Analysis of IgG subclass profiles revealed that IgG1 showed the greatest increasing followed by IgG2b, IgG2a and IgG3. In contrast, IgE level displayed low level of IgE similar to non-immunized mice. In addition, splenocytes of mice immunized with Nfa1 protein secreted significantly high levels of both gamma-interferon and interleukin-10 after stimulation with rNfa1 protein. These findings suggest that the rNfa1 protein may induce the humoral and cell-mediated immunity leading to the host defense in N. fowleri infection.

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TABLE OF CONTENTS

Ⅰ. INTRODUCTION 1
A. Background 1
1. Free-living amoebae 1
2. Naegleri fowleri 2
(A) Life cycle, morphology, growth and culture 3
(B) Pathogenicity 4
(C) Primary amoebic meningoencephalitis 5
(D) Symptoms 7
(E) Diagnosis 7
(F) Treatment 8
(G) Immunity 9
B. Introduction of Naegleria fowleri nfa1gene 12
C. Purpose 12

Ⅱ. MATERIALS AND METHODS 14
A. Culture of Naegleria fowleri trophozoites 14
B. Mice 14
C. Expression and purification of the recombinant His-tag Nfa1 protein 15
D. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 16
E. Immunization 17
1. Intraperitoneal immunization 17
2. Intranasal immunization 18
F. Infection of Naegleria fowleri trophozoites 18
G. Histopathological examination of mice brain tissue infected with N. fowleri trophozoites 19
H. Serum collection 19
I. Antibody detection by Enzyme-linked immunosorbent Assay (ELISA) 20
J. Spleen cell culture 21
K. Cytokines assay 22
L. Duration of the Nfa1- specific IgG levels in immune sera 22

Ⅲ. RESULTS 24
A. Expression and purification of the recombinant His-tag Nfa1 protein 24
B. Survival rate and mean time to death of mice 26
C. Histopathological examination of mice brain tissue infected with N. fowleri trophozoites 29
D. Serum antibody responses to recombinant Nfa1 protein 31
E. Cytokine responses to recombinant Nfa1 protein 38
F. Duration of the Nfa1-specific IgG levels in immunized mice 41

Ⅳ. DISCUSSION 43

Ⅴ. CONCLUSION 48

REFERENCES 50
국문요약 60


LIST OF FIGURES

Fig. 1. SDS-PAGE and Western blotting of the purified Nfa1 protein 25

Fig. 2. The survival rate in immune mice post infection of N. fowleri trophozoites 27

Fig. 3. Histologic findings of mice brain tissues post infection of N. fowleri trophozoites 30

Fig. 4. The Nfa1-specific IgG levels in immune mice sera 33

Fig. 5. Levels of IgG subclass specific the Nfa1 protein in immune mice sera 34

Fig. 6. Levels of IgG subclass specific the Nfa1 protein in immune mice sera 35

Fig. 7. The Nfa1-specific IgA levels in immune mice sera 36

Fig. 8. The Nfa1-specific IgE levels in immune mice sera 37

Fig. 9. Production of IL-2 and IFN-γ from splenocytes of immune mice stimulated with the rNfa1 protein 39

Fig. 10. Production of IL-4 and IL-10 from splenocytes of immune mice stimulated with the rNfa1 protein 40

Fig. 11. Duration of the Nfa1-specific IgG levels in immune sera 42

LIST OF TABLE

Table 1. Mortality of mice infected with N. fowleri trophozoites after they were immunized raperitoneally with the rNfa1 protein in adjuvant or in the absence of adjuvant, or intranasally with the rNfa1 protein only 28

ABBREVIATION

AP: Alkaline Phosphatase
BCIP/NBT: 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium
BSA: Bovine Serum Albumin
CNS: Central Nervous System
ELISA: Enzyme-Linked Immunosorbent Assay
FBS: Fetal Bovine Serum
IFN-γ: Interferon gamma
IL-2: Interleukin-2
IL-4: Interleukin-4
IL-10: Interleukin-10
I.N.: Intranasally
I.P.: Intraperitoneally
IPTG: Isopropylthiogalactoside
OD: Optical Density
PAGE: Polyacrylamide gel electrophoresis
PAME: Primary Amoebic Meningoencephalitis
PBS: Phosphate Buffered Saline
p-NPP: p-Nitrophenyl Phosphate
SDS: Sodium dodecyl sulfate

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