돼지 부종병 예방을 위한 재조합 mStx2e 단백질 백신의 개발 및 평가

목차 (Table of Contents)

• Chapter I. Investigation of pig edema disease antibody and toxin positivity rate in pig farms in Jeollabuk-do, South Korea 1
• I-I Abstract 2
• I-II Introduction 3
• I-III Material and methods 5
• I-III.1 Strains, plasmids, and strain culture conditions used in the experiment 5
• I-III.2 Expression and purification of recombinant Stx2eB -6xHis and mStx2e proteins 8
• I-III.3 Confirmation of purified protein using western blotting 9
• I-III.4 Serum sample used in this study 10
• I-III.5 Determination of optimal conditions for indirect ELISA 11
• I-III.6 Detection of anti-Stx2e antibodies in the serum by indirect ELISA 11
• I-III.7 Monoclonal and polyclonal antibodies against Stx2e 12
• I-III.8 Determination of optimal conditions for sandwich ELISA using absolute quantitative analysis 12
• I-III.9 Measurement of antigen concentration using sandwich ELISA 13
• I-IV Results 14
• I-IV.1 Analysis of purified recombinant mStx2eB and mStx2e protein 14
• I-IV.2 Standard serum for indirect ELISA 17
• I-IV.3 Optimal conditions for indirect ELISA 17
• I-IV.4 Measurement of anti-Stx2e antibody titers using indirect ELISA 18
• I-IV.5 Optimal conditions for sandwich ELISA 24
• I-IV.6 Stx2e toxin detection by sandwich ELISA 25
• I-Ⅴ Discussion 30
• I-VI References 35
• Chapter II. Verification of intracellular movement and function of recombinant eGFP-mStx2e protein 41
• II-I Abstract 42
• II-II Introduction 44
• II-III Material and methods 47
• II-III.1 Strains, plasmids, and strain culture conditions used in the experiment 47
• II-III.2 Expression and purification of recombinant proteins 50
• II-III.3 Cell line used to verify the intracellular transport of recombinant protein 51
• II-III.4 Intracellular tracking of recombinant mStx2e protein with eGFP fluorescence 52
• II-III.5 Intratissue movement of recombinant eGFP-mStx2e protein in mice 53
• II-III.6 Verification of intracellular movement of recombinant mStx2e protein via ICC 54
• II-IV Results 56
• II-IV.1 Analysis of purified recombinant proteins 56
• II-IV.2 Intracellular tracking of recombinant mStx2e protein with eGFP fluorescence 58
• II-IV.3 Physiological responses and tissue distribution following tail vein injection of recombinant eGFP-mStx2e protein in Kunming mice 60
• II-IV.4 Verification of intracellular movement of recombinant mStx2e protein via ICC 62
• II-Ⅴ Discussion 64
• II-VI References 67
• Chapter III. Efficacy evaluation of a recombinant protein vaccine for prevention of porcine edema disease 73
• III-I Abstract 74
• III-II Introduction 76
• III-III Material and methods 79
• III-III.1 Strains, plasmids, and strain culture conditions used in the experiment 79
• III-III.2 Expression and purification of recombinant mStx2e proteins 81
• III-III.3 Confirmation of purified protein using western blotting 82
• III-III.4 Experimental animals used in this study 82
• III-III.5 Experimental design and vaccine administration 83
• III-III.6 Sample collection and antibody assay 86
• III-III.7 Observations and sample collection for safety evaluation of the mStx2e vaccine 86
• III-III.8 Challenge 87
• III-IV Results 90
• III-IV.1 Confirmation of recombinant mStx2e purified protein 90
• III-IV.2 Post-vaccination monitoring 91
• III-IV.3 Safety assessment of the mStx2e vaccine 94
• III-IV.4 Antibody titers 96
• III-IV.5 Monitoring the development of clinical symptoms after challenge and re-isolating the challenge strain 97
• III-IV.6 Necropsy of all surviving piglets 7 days after challenge 100
• III-IV.7 Histopathological examination 102
• III-Ⅴ Discussion 104
• III-VI References 109