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Title Page
Contents
초록 7
Abstract 9
I. Introduction 11
II. Materials and methods 15
1. Bacterial strains and media 15
2. Cell culture 16
3. Determination of efficiency of bacterial uptake and intracellar growth by cultured HeLa cells and macrophages 16
4. LAMP-1 staining 17
5. Bacterial adherence assay 18
6. Labeling bacterial surface proteins 18
7. Virulence determination in mice 19
8. Vaccination against challenge with a virulent strain 19
9. Statistical analysis 19
III. Results 20
1. Morphological characteristic assay 20
2. Bacterial surface protein analysis 21
3. Internalization analysis 22
4. Intracellular replication analysis 22
5. Bacterial adherence activity analysis 25
6. Phagocytic pathway analysis 25
7. Virulence in mice test 26
8. Assay the effects of vaccination with B. abortus △lipA mutant 30
IV. Discussion 31
V. Reference 33
감사의 글 40
Fig. 1. Morphological characteristic of B. abortus wild-type (A) and △lipA mutant (B). Gram's strain. 1000× 20
Fig. 2. Modification of surface proteins of △lipA mutant. Surface proteins of wild-type B. abortus (WT) and △lipA mutant were labeled with Sulfo-NHS-Biotin and then detected by immunoblotting with horseradish peroxidase-conjugated streptividin. The position of... 21
Fig. 3. Bacterial intermalization of △lipA mutant into phagocytes. Wild-type B. abortus (WT) or △lipA mutant was deposited onto HeLa cells(A) and RAW 264.7 cells(B) and then incubated at 37℃ fir the periods of time indicated. Bacterial intermalization efficiency by... 23
Fig. 4. Intercellualr replication of △lipA mutant into phagocytes. HeLa cells(A) and RAW 264.7 cells(B) were infected with wild-type B. abortus (WT) or △lipA mutant as described in Materials and Methods. At different times of incubation, the cells were lysed, and... 24
Fig. 5. Bacterial adherence ability of △lipA mutant on RAW 264.7 cells. Pictures represented bacterial adherence on macrophages (A), (B) was the number of adherentic bacteria per 100 cells, Date were shown the averages of triplicate samples from three identical... 27
Fig. 6. LAMP-1 colocalization on bacteria containing phagosomes. RAW 264.7 cells were infected with wild-type B. abortus (WT) or △lipA mutant, and incubated for 30 min, fixed, and stained for LAMP-1. The samples were visualized by immunofluorescence... 28
Fig. 7. Virulence of △lipA mutant in mice. Mice were infected intrapeitoneally with control or △lipA mutant (10⁴ CFU/0.1㎕). Recovery of variable bacteria from the spleen(A) and the weights of spleen(B) of infected mice at 2~8 weeks postinfection are shown.... 29
Fig. 8. Protection against B. abortus 544 in mice vaccinated with △lipA mutant. The mice were inoculated intraperitoneally with 10⁴ CFU of mutant. After six weeks vaccination, mice were challenged with B. abortus 544. Two weeks later, mice were killed and the numbers... 30
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