Construction of recombinantly attenuated bacterial vaccines and their use for development of combined vaccine system
- Alternative Title
- 재조합 약독화 세균 백신 제작 및 이를 기반으로 하는 복합 백신 시스템 개발
- Abstract
- The aims of the present study were development of live attenuated bacterial vaccines and further assessment of potential of the attenuated bacteria as vehicles for combined vaccines. To achieve the goals, firstly, we generated an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) by knock-out of two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis - alanine racemase gene (alr) and aspartate semialdehyde dehydrogenase gene (asd). Virulence of the E. tarda mutant in olive flounder (Paralichthys olivaceus) was highly decreased, which was demonstrated by approximately 106 fold increase of LD50 dose compared to wild-type E. tarda. Immunization of fish with the auxotrophic E. tarda mutant through either intraperitoneal (i.p.) or oral routes induced significantly higher serum agglutination activities and clearly higher survival rates against E. tarda challenges. Secondly, to generate auxotrophic E. tarda mutant that has no need for supplementation of specific nutrients into culture medium, we newly constructed an E. tarda mutant by replacement of alr gene promoter with cI857-λPR promoter system plus another cI857 expression cassette that was driven by a constitutive promoter of E. tarda (EtPR C28-1), which allow the mutant bacteria to grow at temperature above 30°C without supplement of D-alanine but to disintegrate below 30°C. In vaccine experiment, olive flounder fingerlings immunized with the temperature-sensitive mutant E. tarda showed greatly decreased mortality, and a boost-immunization induced complete protection against E. tarda infection. Thirdly, to evaluate the potential of the auxotrophic E. tarda mutant as a delivery vehicle for a heterologous antigen, the mutant bacteria was transformed with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression, which induced significantly higher ELISA titer against GFP antigen in olive flounder by i.p. immunization. Fourthly, to further evaluate potential of the Δalr Δasd E. tarda as a delivery vehicle for DNA vaccine in fish, olive flounder were immunized with the E. tarda mutant harboring plasmids for CMV promoter-driven eGFP, which was successful to express the antigen in the internal organs and to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter antigen of DNA vaccine. Furthermore, fish immunized with the mutant E. tarda harboring plasmids for marine medaka β-actin promoter-driven VHSV G gene showed significantly higher serum neutralization activity and higher survival rates against VHSV challenges. The present results indicate that auxotrophic mutants of fish pathogenic bacteria are effective candidates for fish vaccines and can be used as vehicles for delivery of heterologous antigens or DNA vaccine plasmids.
- Issued Date
- 2012
- Awarded Date
- 2012. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Alternative Author(s)
- 최승혁
- Affiliation
- 수산생명의학과
- Department
- 대학원 수산생명의학과
- Advisor
- 김기홍
- Table Of Contents
- CONTENTS
Abstract ii ⅴ
List of Tables ix
List of Figures x
General introduction 1
Chapter I. Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus) 5
Introduction 6
Materials and methods 10
1. Bacterial strains and culture conditions 10
2. Fish 10
3. Generation of alanine racemase gene (alr) and aspartate semialdehyde dehydrogenase (asd) gene knock-out E. tarda 10
4. Construction of antibiotic resistance gene-free and heterologous gene-expressing vector 12
5. In vitro growth of recombinant E. tarda 13
6. Determination of LD50 of Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP 14
7. In vivo persistence of Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP 14
8. Vaccination and challenge 15
9. Agglutination activity of serum against E. tarda 17
10. ELISA 17
11. Western blot 18
12. Statistical analyses 19
Results 21
1. Generation of alanine racemase gene (alr) and aspartate semialdehyde dehydrogenase gene (asd) knock-out E. tarda (Δalr Δasd E. tarda) 21
2. Construction of plasmid pG02-ASD-EtPR-GFP 23
3. Virulence and persistence of Δalr Δasd E. tarda in olive flounder (Experiment 1) 26
4. Vaccine efficacy of Δalr Δasd E. tarda (Experiment 1) 28
5. Serum agglutination activity, ELISA, and Western blot (Experiment 1) 30
6. Persistence of orally administered Δalr Δasd E. tarda in olive flounder (Experiment 2) 35
7. Protection of fish by oral immunization with Δalr Δasd E. tarda (Experiment 2) 35
8. Agglutination activity, ELISA, and Western blot (Experiment 2) 37
Discussion 39
Chapter II. Generation of a temperature-sensitive Edwardsiella tarda mutant and its potential as a prophylactic vaccine in olive flounder (Paralichthys olivaceus) 44
Introduction 45
Materials and methods 47
1. Bacterial strains and culture 47
2. Generation of temperature-sensitive E. tarda mutant by replacement of alr gene promoter with a temperature-sensitive promoter system 47
3. In vitro growth of temperature-sensitive E. tarda mutant 48
4. Semi-quantitative RT-PCR analysis 48
5. In vivo virulence of temperature-sensitive E. tarda mutant 49
6. In vivo persistence of temperature-sensitive E. tarda mutant 49
7. Determination of protective efficacy of temperature-sensitive E. tarda mutant 50
8. Serum agglutination activity 50
9. Statistical analysis 51
Results 53
1. Generation of temperature-sensitive E. tarda mutant 53
2. Semi-quantitative RT-PCR analysis to measure alr gene transcription 56
3. In vivo virulence and persistence of temperature-sensitive E. tarda mutant 58
4. Vaccine efficacy of temperature-sensitive E. tarda mutant and agglutination activity of serum 61
Discussion 64
Chapter III. Potential of auxotrophic Edwardsiella tarda double-knockout mutant as a delivery vector for DNA vaccine in olive flounder (Paralichthys olivaceus) 67
Introduction 68
Materials and methods 71
1. Bacterial strains and culture conditions 71
2. Vector construction for DNA vaccine 71
3. In vivo persistence of orally administered Δalr Δasd E. tarda 74
4. In vivo administration of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP 74
5. Western blot analysis to confirm the expression of eGFP in internal organs 74
6. Serum agglutination activity against E. tarda 75
7. Western blot analysis to confirm serum antibody against eGFP 76
Results 77
1. Persistence of orally administered Δalr Δasd E. tarda in olive flounder 77
2. Expression of EGFP in internal organs 79
3. Serum agglutination activity against E. tarda 82
4. Generation of serum antibody against eGFP 83
Discussion 84
Chapter IV. Protection of olive flounder (Paralichthys olivaceus) from viral hemorrhagic septicemia virus (VHSV) by immunization with auxotrophic Edwardsiella tarda mutant harboring VHSV DNA vaccine 86
Introduction 87
Materials and methods 89
1. Bacterial strains and culture conditions 89
2. Oryzias dancena β-actin gene 89
3. Cells culture, virus and transfection 89
4. Construction of heterologous expression vector 90
5. In vitro RFP expression assay 91
6. In vivo intramuscular injection RFP expression assay 91
7. Vaccination and challenge 92
8. Neutralization test 92
9. Statistical analyses 93
Results 95
1. Vector Construction of DNA vaccine 95
2. In vitro & in vitro RFP expression of compared the power promoter 97
3. Protective efficacy against VHSV challenges 100
4. Serum neutralization activity 102
Discussion 103
Summary 106
Abstract in Korean 111
Acknowledgment 113
References 115
- Degree
- Doctor
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