论文说明：List of figures、list of Tables
DECLARATION
Acknowledgements
摘要
Introduction
Chapter 1 - Rabbit hemorrhagic disease and its causative agent
Section A Rabbit hemorrhagic disease: An introduction
Section B Rabbit hemorrhagic disease virus: structure, genome and variants
Chapter 2 - VP60 capsid protein: Structure and expression as subunit vaccine in heterologous systems
2.1 RHDV capsid structure
2.2 Expression of VP60 Capsid protein as subunit vaccine in heterologous systems
References
3.1 Design of primers
3.2 Isolation of total RNA from RHDV-infected livers
3.3 Reverse transcriptase-polymerase chain reaction (RT-PCR)
3.4 Cloning of VP60 gene fragments into pUC 19 plasmid
3.4.1 Preparation of inserts
3.4.2 Preparation of the pUC19 plasimd for ligation
3.4.3 Ligation of insert DNAs with pUC 19 plasmid DNA
3.4.4 Transformation of competent E.coli DH5α cells with pUC constructs
3.5 Results
3.6 Discussion
CHAPTER 4-Expression of Full and Partial-length RHDVVP60 Gene Fragments in E.coli
4.1 Overview of pET Expression System
4.2 Cloning of VP60 gene fragments into pET-30a
4.2.1 Preparation of VP60 gene inserts
4.2.2 Preparation of the pET30-a plasimd for ligation
4.2.3 Ligation of insert DNAs with pET-30a plasmid DNA
4.2.4 Transformation of competent E. coli cells with pET constructs
4.3 Expression of full and partial-length RHDV VP60 Proteins in E. coli: induction, identification and purification
4.3.1 Optimization of expression conditions for recombinant proteins
4.3.2 Identification of proteins by SDS-polyacrylamide gel electrophoresis(SDS-PAGE)
4.3.3 Expression and purification of His-tagged proteins;VP60-A,VP60-B and VP60-C
4.3.4 Regeneration ofNi-NTA agarose beads
4.4 Results
4.5 Discussion
Chapter 5- Immunoreactivity of full and partial-length RHDV VP60 Proteins
5.1 Raising RHDV anti-serum
5.2 Determination of protein concentrations
5.3 Testing the immunoreactivity of VP60 proteins and development of an ELISA for measuring antibody response to vaccination
5.3.1 Enzyme-linked immunosorbent assay (ELISA)
5.3.2 Western blotting
5.4 Evaluation of ELISA using the field serum samples
5.5 Results
5.6 Discussion
Chapter 6 - Expression of full and Partial-length RHDV VP60 gene fragments in Sf9 insect cell line.
6.1 Overview of Bac-to-Bac baculovirus expression system
6.2 Cloning of VP60 gene fragments into pFastBac HT A plasmid
6.2.1 Preparation of inserts
6.2.2 Preparation of the pFastBac HT A plasimd for ligation
6.2.3 Ligation of insert DNAs with pFastBac HT A plasmid DNA
6.2.4 Transformation of competent E. coli cells with pFastBac constructs
6.3 Generation of recombinant baculoviruses and protein expression
6.3.1 Generation the recombinant bacmids
6.3.2 Production of recombinant baculoviruses
6.3.3 Expressing the recombinant proteins
6.4 Testing the immunoreactivity of VP60 proteins
6.4.1 Western blot analysis
6.4.2 Immunofluorescence assay
6.5 VLPs preparation and electron microscopy
6.6 Results
6.7 Discussion
Conclusion
References
