by Lau On Sun. / Thesis submitted in: November 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 117-127). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Abbreviations --- p.viii / Table of Contents --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global situation --- p.3 / Chapter 2.1.2 --- Malaria parasite and its life cycle --- p.4 / Chapter 2.1.3 --- Need for a malarial vaccine --- p.5 / Chapter 2.2 --- Merozoite surface protein 1 and its fragments - the advanced malaria vaccine candidate --- p.7 / Chapter 2.2.1 --- Basic research on MSP1 --- p.7 / Chapter 2.2.2 --- Vaccine research on MSP1 --- p.8 / Chapter 2.3 --- Transgenic plants as recombinant protein production systems --- p.11 / Chapter 2.3.1 --- Characteristics --- p.11 / Chapter 2.3.2 --- Plant-based vaccine --- p.13 / Chapter 2.4 --- Expression of MSP 1 C-terminal fragments in transgenic plants --- p.15 / Chapter 2.4.1 --- Previous studies --- p.15 / Chapter 2.4.2 --- Plant-optimized MSP142 cDNA --- p.18 / Chapter 2.5 --- Phaseolin: its promoter and vacuolar-sorting signal --- p.20 / Chapter 2.6 --- Sorting of soluble protein to vacuoles in plants --- p.22 / Chapter 2.7 --- Winged bean lysine-rich protein and translational fusion strategy --- p.24 / Chapter 2.8 --- Hypotheses and aims of study --- p.26 / Chapter Chapter 3: --- Materials and Methods --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Chemicals --- p.28 / Chapter 3.3 --- Bacterial strains --- p.28 / Chapter 3.4 --- Chimeric gene construction --- p.29 / Chapter 3.4.1 --- Construction of the lysine-rich protein fusion constructs --- p.33 / Chapter 3.4.2 --- Construction of the phaseolin-targeting constructs --- p.37 / Chapter 3.4.3 --- Confirmation of sequence fidelity of chimeric genes --- p.42 / Chapter 3.4.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.42 / Chapter 3.5 --- Transgenic expression in Arabidopsis and tobacco --- p.44 / Chapter 3.5.1 --- Plant materials --- p.44 / Chapter 3.5.2 --- Agrobacterium transformation --- p.44 / Chapter 3.5.3 --- Arabidopsis transformation and selection --- p.45 / Chapter 3.5.4 --- Tobacco Transformation and Selection --- p.47 / Chapter 3.5.5 --- Genomic DNA isolation --- p.49 / Chapter 3.5.6 --- Southern blot analysis --- p.49 / Chapter 3.5.7 --- Total silique RNA isolation --- p.50 / Chapter 3.5.8 --- Northern blot analysis --- p.50 / Chapter 3.5.9 --- Protein extraction and SDS-PAGE --- p.51 / Chapter 3.5.10 --- Western blot analysis --- p.52 / Chapter 3.5.11 --- Enterokinase digestion of recombinant LRP fusion protein --- p.53 / Chapter 3.5.12 --- Deglycosylation studies of recombinant MSP142-AFVY --- p.54 / Chapter 3.6 --- Confocal immunoflorescence studies of MSPl42-AFVY in tobacco --- p.55 / Chapter 3.6.1 --- Preparation of sections --- p.55 / Chapter 3.6.2 --- Labeling of fluorescence probes --- p.55 / Chapter 3.6.3 --- Image collection --- p.56 / Chapter 3.7 --- Bacterial expression of MSP 142 and anti-serum production --- p.57 / Chapter 3.7.1 --- pET expression in E. coli --- p.57 / Chapter 3.7.2 --- Purification of recombinant His-MSPl42 --- p.58 / Chapter 3.7.3 --- Immunization of rabbits --- p.59 / Chapter Chapter 4: --- Results --- p.60 / Chapter 4.1 --- Transgenic analysis of lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.1 --- Construction of the lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.2 --- Selection of transgenic plants --- p.62 / Chapter 4.1.3 --- Southern analysis --- p.65 / Chapter 4.1.4 --- Northern analysis --- p.69 / Chapter 4.1.5 --- Western analysis --- p.71 / Chapter 4.1.6 --- Western analysis with anti-LRP --- p.75 / Chapter 4.1.7 --- Enterokinase digestion of recombinant LRP fusion protein --- p.76 / Chapter 4.2 --- Transgenic analysis of phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.1 --- Construction of the phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.2 --- Selection of transgenic plants --- p.82 / Chapter 4.2.3 --- Southern analysis --- p.85 / Chapter 4.2.4 --- Northern analysis --- p.89 / Chapter 4.2.5 --- Western analysis --- p.91 / Chapter 4.2.6 --- Deglycosylation studies of recombinant MSPl42-AFVY --- p.96 / Chapter 4.2.7 --- Human serum detection of MSP142-AFVY --- p.100 / Chapter 4.3 --- Confocal immunofluorescence studies of MSP142-AFVY in tobacco --- p.102 / Chapter 4.4 --- Bacterial expression of MSPl42 and anti-serum production --- p.105 / Chapter 4.4.1 --- Expression and purification of recombinant His-MSPl42 in E. coli --- p.105 / Chapter 4.4.2 --- Titer and specificity of the anti-serum --- p.107 / Chapter Chapter 5 --- Discussion --- p.109 / Chapter Chapter 6 --- Conclusion --- p.116 / References --- p.117
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324796 |
| Date | January 2004 |
| Contributors | Lau, On Sun., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xv, 127 leaves : ill. ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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