Title Page
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATIONS 10
ABSTRACT 11
I. Introduction 12
I.1. Mucosal vaccination 12
I.2. Lactic acid bacteria as a mucosal vaccine delivery vector 13
I.2.1. Lactic acid bacteria 14
I.2.2. Lactococcus lactis as live vehicles 14
I.2.3. Gene expression systems for lactic acid bacteria 15
I.3. Human papillomavirus (HPV) 15
I.3.1. Human papillo mavirus type16 15
I.3.2. Virus-like particles (VLPs) 16
I.4. Purpose of this study 16
II. Materials and methods 18
II.1. Bacterial strains, plasmids and growth conditions 18
II.2. DNA manipulation 18
II.3. Plasmid constructions 18
II.4. RNA isolation and reverse transcription polymerase chain reaction analysis (RT-PCR) 21
II.5. Preparation of intracellular and supernatant fractions of L. lactis and immunoblotting 22
II.6. Animals 23
II.7. Oral immunization of mice 24
II.8. Sample collection 24
II.9. Preparation of HPV-16 L1 VLP 26
II.10. Evaluation of antibody production 26
III. Results 28
III.1. Lactic acid-producing and growth capability test of L. lactis producing HPV-16 L.1 28
III.2. The intracelluar form and secreted forms of L.1 were stably produced in L. lactis 31
III.3. Serum antibody responses to HPV-16 L.1 expressed by L. lactis 34
III.4. Vaginal antibody responses to HPV-16L1expressed by L. lactis 38
III.5. Intestinal antibody responses to HPV-16 L1 expressed by L. lactis 38
IV. Discussion 42
REFERENCES 44
SUMMARY IN KOREAN 50
Table 1. Bacterial strains and plasmids used in this study 19
Table 2. Vaccination protocols 25
Figure1. Schematic representation of two expression vectors 20
Figure2. Growth curves of different strains 29
Figure3. Lactic acid-producing capability test 30
Figure4. L1 transcription pattern in L. lactis 32
Figure5. L1 expression in L. lactis 33
Figure6. Systemic immune responses after oral administration 36
Figure7. Systemic immune responses afte rora ladministration 37
Figure8. Mucosal immune responses after oral administration 40
Figure9. Mucosal immune responses after oral administration 41