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Title page
Abstract
Contents
Chapter I. General introduction 18
1.1. Introduction 19
1.2. History 22
1.3. Structure of BoNTs complexes 24
1.4. Intoxication mechanism of BoNTs 27
1.4.1. Extracellular cell binding 27
1.4.2. Internalization 28
1.4.3. Membrane translocation 28
1.4.4. Metalloprotease activity 31
1.5. Vaccines and neutralizing antibodies against BoNTs 33
1.6. Overview of BoNT/A light chain and biochemical activity. 34
1.7. Objectives of this research 35
Chapter II. Materials and methods 37
2.1. Materials 38
2.1.1. Chemicals, Buffers and Reagents 38
2.1.2. Bacterial strains, plasmids and purification systems 38
2.1.3. Botulinum neurotoxins 38
2.2. Methods 39
2.2.1. Preparation of BoNT/F Toxoid 39
2.2.2. Toxoid immunization 39
2.2.3. Preparation of monoclonal antibody against BoNT/F 39
2.2.4. Indirect enzyme linked immunosorbent assay (ELISA) 40
2.2.5. Western blot analysis 41
2.2.6. Immunoprecipitation assay 43
2.2.7. In vivo neutralization assay 43
Chapter III. Isolation and characterization of a neutralizing antibody specific to Clostridium botulinum neurotoxin serotype F 44
3.1. Background 45
3.2. Results and discussion 47
3.2.1. Isolation of monoclonal antibody specific to BoNT/F 47
3.2.2. Indirect ELISA 47
3.2.3. Western blot analysis 52
3.2.4. Purification of FHNM monoclonal antibody 52
3.2.5. Immunoprecipitation 54
3.2.6. Neutralization activity 56
Chapter IV. Biochemical analysis of Clostridium botulinum type A light chain (BoNT/A LC) and its mutants. 59
4.1. Background 60
4.2. Materials and methods 63
4.2.1. rBoNT/A LC, Chemicals, Buffers and Reagents 63
4.2.2. Site directed mutagenesis 63
4.2.3. Expression and purification of GST-A/LC (Wild type) and its mutants 69
4.2.4. Endopeptidase Assay 69
4.2.5. Autocatalysis assay 70
4.3. Results and discussion 70
4.3.1. Site-directed mutagenesis 70
4.3.2. Expression and purification of GST-A/LC (wild type) and its mutants 71
4.3.3. Endopeptidase activity 71
4.3.4. Autocatalytic activity 76
Chapter V. Cloning, expression, purification, and characterization of the partial fragments of BoNT/F 81
Abstract 82
5.1. Background 83
5.2. Methods 84
5.2.1. Construction of BoNT/F partial fragments 84
5.2.2. Expression and purification of the partial fragments proteins 85
5.2.3. Production and analysis of polyclonal antibodies from the mice immunized with recombinants FLC and FHN proteins. 85
5.3. Results and discussion 89
5.3.1. Construction of BoNT/F different Fragments 89
5.3.2. Expression and purification of partial recombinant fragments 92
5.3.3. Production and analysis of polyclonal antisera from the mice immunized with recombinants FLC and FHN proteins. 93
Conclusions and future prospective 98
References 100
Acknowledgements 105
국문요약 107
Table 1.1. TeNT-and BoNT-producing clostridia, neurotoxin gene localization, and neurotoxin amino acid length. 21
Table 3.1. In vivo neutralization assay using FHNM ascite fluid. 57
Table 3.2. In vivo neutralization assay using purified FHNM mAb. 58
Table 4.1. Primers used for the alanine mutagenesis of nine amino acid residues near and within active site of BoNT/A LC by site-directed mutagenesis. 68
Table 5.1. Primers used for the amplification of BoNT/F partial fragments. 88
Fig. 1.1. Structure of botulinum toxin and toxin complexes 26
Fig. 1.2. Toxicity mechanism of BoNT. 30
Fig. 1.3. Molecular targets of clostridial neurotoxins. 32
Fig. 2.1. Flow Chart of the hybridoma technique for the production of the monoclonal antibody specific to BoNT/F. 42
Fig. 3.1. Indirect ELISA performed using BoNT/F as a coating antigen. 49
Fig. 3.2. Indirect ELISA performed using BoNT/A, -/E, -/F, and BSA as coating antigens. 50
Fig. 3.3. Indirect ELISA performed using rFLC, rFHN, and rFHCc as coating antigens. 51
Fig. 3.4. Purification of mAb FHNM on Protein G-Sepharose affinity column. 53
Fig. 3.5. Characterazation of the epitope of mAb FHNM by Immuno-precipitation assay. 55
Fig. 4.1. Proposed hydrolytic mechanism of active site residues of BoNT/A LC 62
Fig. 4.2. BoNT/A LC wild type sequence and site-directed alanine mutagenesis strategy. 67
Fig. 4.3. Analysis of proteolytic activities of BoNT/A LC (wild type) and its alanine mutants by endopeptidase assay. 74
Fig. 4.4. Analysis of proteolytic activity of wild type A/LC and its mutants performing the endopeptidase assay 5 h at 37 C. 75
Fig. 4.5. Analysis of autocatalytic activities of BoNT/A LC (wild type) and its mutants reacted 1week on glycerol at R/T. 78
Fig. 4.6. Analysis of biochemical activities of BoNT/A LC and its mutants by autocatalysis performed 1 week at R/T. 79
Fig. 4.7. Analysis of biochemical activities of BoNT/A LC and its mutants by the autocatalysis performed 3 h in the presence of Zn at 37 C. 80
Fig. 5.1. Cloning strategy of partial fragments of BoNT/F. 87
Fig. 5.2. Agarose gel electrophoresis of PCR and plasmid digestion of rFLC and rFHN. 90
Fig. 5.3. Agarose gel electrophoresis of PCR and plasmid digestion of rFHC and rFHCc. 91
Fig. 5.4. Purification and characterization of rFLC. 94
Fig. 5.5. Purification and characterization of rFHN. 95
Fig. 5.6. Indirect ELISA for the analysis of immunoreactivity against rFLC. 96
Fig. 5.7. Indirect ELISA test for the analysis of immunoreactivity against rFHN. 97
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