| Abstract | 第5-6页 |
| 摘要 | 第7-12页 |
| Abbreviations | 第12-14页 |
| CHAPTRE I Literature review | 第14-43页 |
| 1.1 Introduction | 第14-15页 |
| 1.2 Bluetongue | 第15-25页 |
| 1.2.1 History and current global distribution of the disease | 第15-18页 |
| 1.2.2 The disease transmission | 第18-21页 |
| 1.2.3 The disease epidemiology | 第21-23页 |
| 1.2.4 The pathogenesis of bluetongue virus | 第23-25页 |
| 1.3 Bluetongue virus | 第25-32页 |
| 1.3.1 The outer most proteins VP2 and VP5 | 第27-28页 |
| 1.3.2 Structural organization of BTV core and associated proteins | 第28页 |
| 1.3.3 Non-structural proteins | 第28-30页 |
| 1.3.4 Replication cycle of BTV | 第30-32页 |
| 1.4 Vaccine development | 第32-41页 |
| 1.4.1 Bluetongue vaccination strategies | 第33-34页 |
| 1.4.2 Types of BTV vaccines | 第34-41页 |
| 1.4.2.1 Conventional live and inactivated vaccines | 第35-36页 |
| 1.4.2.2 Recombinant viral vector vaccines | 第36-37页 |
| 1.4.2.3 Recombinant subunit vaccines | 第37-39页 |
| 1.4.2.4 DNA vaccines | 第39-40页 |
| 1.4.2.5 Reverse genetics-based vaccines | 第40-41页 |
| 1.5 Aims of the current study | 第41-43页 |
| CHAPTER II. Expression of full-length BTV proteins in baculovirus expression system | 第43-58页 |
| 2.1 Introduction | 第43-44页 |
| 2.2 Materials and methods | 第44-50页 |
| 2.2.1 Virus, genes and cells | 第44页 |
| 2.2.2 PCR, electrophoresis and DNA purification | 第44-46页 |
| 2.2.3 Cloning into pFastBac? HTb vector | 第46页 |
| 2.2.4 Transposition of recombinant pFastBac?HTb into the bacmid DNA | 第46-47页 |
| 2.2.5 Isolation of recombinant plasmid and bacmid DNA | 第47页 |
| 2.2.6 Transfection of Sf9 cells with recombinant bacmid for production of the recombinant baculovirus | 第47-49页 |
| 2.2.7 Expression and identification of recombinant BTV proteins | 第49页 |
| 2.2.8 Purification and quantification of recombinant proteins | 第49-50页 |
| 2.3 Results | 第50-55页 |
| 2.3.1 Construction of recombinant p Fast Bac?HTb vectors | 第50-51页 |
| 2.3.2 Generating the recombinant bacmids | 第51-52页 |
| 2.3.3 Transfection of Sf9 cells and identification of the expressed proteins | 第52-53页 |
| 2.3.4 Protein expression and purification | 第53-55页 |
| 2.4 Discussion | 第55-58页 |
| CHAPTER III Prokaryotic expression of full-length BTV-16VP3, VP5, VP7, NS2, and truncatedversion of VP5 | 第58-69页 |
| 3.1 Introduction | 第58-59页 |
| 3.2 Materials and methods | 第59-62页 |
| 3.2.1 Virus genes and cells | 第59页 |
| 3.2.2 Construction of the recombinant expression plasmids | 第59页 |
| 3.2.3 Expression and identification of BTV-16 proteins | 第59-61页 |
| 3.2.4 Purification and quantification of the expressed proteins | 第61-62页 |
| 3.2.5 Assembly of core like particles | 第62页 |
| 3.3 Results | 第62-66页 |
| 3.3.1 Construction of recombinant bacterial expression plasmids | 第62页 |
| 3.3.2 Prokaryotic expression and identification of BTV proteins | 第62-65页 |
| 3.3.3 Protein purification and quantification | 第65页 |
| 3.3.4 Assembly and characterization of BTV CLPs | 第65-66页 |
| 3.4 Discussion | 第66-69页 |
| CHAPTER IV Immunogenicity evaluation of protein-based subunit vaccines against bluetonguevirus in BALB/C mice and sheep | 第69-85页 |
| 4.1 Introduction | 第69-70页 |
| 4.2 Materials and methods | 第70-73页 |
| 4.2.1 Ethics statement | 第70页 |
| 4.2.2 Cell lines, viruses and animals | 第70页 |
| 4.2.3 Animal immunization | 第70-71页 |
| 4.2.3.1 Immunization of sheep | 第70-71页 |
| 4.2.3.2 Immunization of BALB/c | 第71页 |
| 4.2.4 Serological analyses | 第71-73页 |
| 4.2.4.1 Detection of BTV-specific antibodies by cELISA | 第71-72页 |
| 4.2.4.2 Detection of protein-specific antibodies by indirect ELISA | 第72页 |
| 4.2.4.3 Plaque reduction neutralization assay (PRNA) | 第72-73页 |
| 4.2.4.4 Antigen specific lymphocyte proliferation assay in sheep | 第73页 |
| 4.3 Results | 第73-81页 |
| 4.3.1 Immunogenicity of the recombinant subunit vaccines in sheep | 第73-74页 |
| 4.3.2 Immunogenicity of the recombinant subunit vaccines in mice | 第74-79页 |
| 4.3.3. Detection of protein-specific antibodies | 第79-80页 |
| 4.3.4 Plaque reduction neutralization assay | 第80-81页 |
| 4.3.5 Lymphocyte proliferation responses | 第81页 |
| 4.4 Discussion | 第81-85页 |
| Conclusion | 第85-86页 |
| References | 第86-104页 |
| Acknowledgements | 第104-105页 |
| Curriculum Vitae | 第105-106页 |
