| 摘要 | 第1-12页 |
| Abstract | 第12-14页 |
| 1 Introduction | 第14-21页 |
| ·Infectious Bursal Disease | 第14-17页 |
| ·Clinical Findings of IBD | 第14-15页 |
| ·VP2 as an antigenic component | 第15-16页 |
| ·Vaccinations progress against IBD | 第16-17页 |
| ·Lactobacillus an attractive expression system | 第17-19页 |
| ·Bacteriophage T7 gene 10 derived (RBS) translational enhancer | 第17-18页 |
| ·Electro-transformation efficient tool for plasmid DNA uptake | 第18-19页 |
| ·Advantages of mucosal immunization | 第19页 |
| ·Objective of this study | 第19-21页 |
| 2 Materials and Methods | 第21-37页 |
| ·Materials | 第21-24页 |
| ·Bacterial strains and plasmids | 第21页 |
| ·Virus | 第21页 |
| ·Experimental Animals | 第21页 |
| ·Antibodies and Test Kits | 第21页 |
| ·Primers | 第21-22页 |
| ·Chemical reagents | 第22页 |
| ·Medium and antibiotic | 第22页 |
| ·Instrument and equipment | 第22-24页 |
| ·Method | 第24-37页 |
| ·Cloning and sequencing of vp2 gene | 第24页 |
| ·Activation of bacteria | 第24页 |
| ·Vp2 gene amplification | 第24-25页 |
| ·Gene extraction and ligation of vp2 with p MD18-T simple vector | 第25-26页 |
| ·Heat shock transformation method | 第26页 |
| ·Screening and identification of recombinant strains | 第26-27页 |
| ·Engineering of expression vector | 第27-30页 |
| ·Preparation of competent cells and bacterial electroporation | 第30-33页 |
| ·Expression of recombinant protein and identification by SDS-PAGE and Western blot | 第33-34页 |
| ·Identification of vp2 surface expression by immunofluorescence | 第34页 |
| ·Propagation of IBDV | 第34-35页 |
| ·Immunization | 第35页 |
| ·Determination of antibody by ELISA | 第35-36页 |
| ·Detection of lymphocytes proliferation | 第36页 |
| ·Statistical analysis | 第36-37页 |
| 3 Results | 第37-57页 |
| ·Vp2 amplification and cloning results | 第37-38页 |
| ·PCR amplification result of target vp2 gene | 第37页 |
| ·Recombinant plasmid p MD18-TS-vp2 result | 第37-38页 |
| ·Confirmation result of p MD18-TS-vp2 by single and double digestion | 第38页 |
| ·Construction and identification result of recombinant expression vector | 第38-44页 |
| ·Engineering of expression vector | 第38-40页 |
| ·Identification of recombinant expression plasmids by enzyme digestion and PCR assay | 第40-42页 |
| ·Electrotransformation result of recombinant expression plasmid into Lactobacillus strains | 第42-43页 |
| ·Comparison of old and improved method | 第43-44页 |
| ·immunofluorescence results of vp2 surface expression | 第44-47页 |
| ·SDS-PAGEand Western blot result | 第47-49页 |
| ·Antibody titire and stimulation of humoral immune response | 第49-51页 |
| ·Detection of Th1 and Th2 type immune response | 第51-56页 |
| ·Lymphocytes proliferation | 第51-54页 |
| ·Detection of cytokines-based immune response | 第54-56页 |
| ·Protective efficacy against IBDV challenge | 第56-57页 |
| 4 Discussion | 第57-62页 |
| ·Competent cells preparation of Lactobacillus strains | 第57-58页 |
| ·DNA-based vaccines induce cellular and humoral immune responses | 第58-59页 |
| ·Expression of vp2 protein on the cell surface using HCE-Pgs A system | 第59页 |
| ·Comparison of vp2 protein expression using translational enhancer T7g10 | 第59-60页 |
| ·Humoral immune response against IBDV | 第60页 |
| ·Cellular immune response mediated by T cells | 第60页 |
| ·Th1 and Th2 cytokines immune responses | 第60-62页 |
| 5 Conclusions | 第62-63页 |
| Acknowledgement | 第63-64页 |
| References | 第64-68页 |
| Appendix | 第68-70页 |
| Papers published in the periods of Ph.D. education | 第70页 |
