Yang, Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-132). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / LIST OF ABBREVIATIONS --- p.xvi / Chapter Chatper 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.5 / Chapter 2.1 --- Facts on food allergy --- p.5 / Chapter 2.1.1 --- Food allergy and its prevalence --- p.5 / Chapter 2.1.2 --- Pathogenesis of food allergy --- p.6 / Chapter 2.1.3 --- Clineal disorders caused and diagnosis of food allergy --- p.8 / Chapter 2.2 --- Allergenicity assessment of genetically engineered food --- p.13 / Chapter 2.2.1 --- The structural and sequence homology of proteins as a criterion for food allergenicity assessment --- p.14 / Chapter 2.2.2 --- Digestion stability as a criterion for food allergenicity assessment --- p.15 / Chapter 2.2.3 --- Animal models for Food Allergenicity Assessment --- p.21 / Chapter 2.3 --- The importance of rice and its nutritional facts --- p.27 / Chapter 2.3.1 --- The importance of rice --- p.27 / Chapter 2.3.2 --- Rice nutritional facts and its relationship with malnutrition --- p.28 / Chapter 2.4 --- Food allergenicity research in rice --- p.30 / Chapter 2.5 --- Glutelin overexpression transgenic rice GT3 --- p.33 / Chapter 2.6 --- Recent and future perspectives for treatment of food allergy --- p.36 / Chapter Chapter 3. --- Materials and Methods --- p.39 / Chapter 3.1 --- Rice Seed Protein Extraction --- p.39 / Chapter 3.1.1 --- Rice varieties for protein extraction --- p.39 / Chapter 3.1.2 --- Protein extraction from rice seeds --- p.39 / Chapter 3.1.3 --- Fractionation of major rice seed storage proteins --- p.40 / Chapter 3.1.4. --- Protein quantification --- p.41 / Chapter 3.1.5 --- Tricine SDS-PAGE --- p.42 / Chapter 3.2 --- Simulated Gastric Digestibility Assay --- p.43 / Chapter 3.2.1 --- Assay System --- p.43 / Chapter 3.2.2 --- Preparation of Simulated Gastric Fluid --- p.43 / Chapter 3.2.3 --- Assay Procedures --- p.44 / Chapter 3.2.4 --- Results Interpretation --- p.44 / Chapter 3.3 --- Construction of Mouse Models --- p.45 / Chapter 3.3.1 --- Mouse strain and reagents used --- p.45 / Chapter 3.3.2 --- Mouse Model I --- p.46 / Chapter 3.3.3 --- Mouse Model II --- p.50 / Chapter 3.3.4 --- Mouse Model III --- p.51 / Chapter 3.4 --- Bioinformatic Analysis of Glutelin Sequence --- p.52 / Chapter 3.5 --- Epitope Mapping of Glutelin --- p.55 / Chapter 3.5.1 --- Bioinformatic Analysis --- p.55 / Chapter 3.5.2 --- Direct and Competitive ELISA --- p.56 / Chapter 3.5.3 --- Western Blot Analysis --- p.57 / Chapter 3.5.4 --- IgE-binding assay --- p.58 / Chapter Chapter 4. --- Results and Discussion --- p.60 / Chapter 4.1 --- Rice Seed Protein Extraction --- p.60 / Chapter 4.1.1 --- Rice Protein Extraction --- p.60 / Chapter 4.1.2 --- Extraction of rice major seed storage protein fractions --- p.62 / Chapter 4.2 --- Simulated Gastric Digestibility Assay --- p.64 / Chapter 4.2.1 --- Pepsin Digestibility of total protein from GT3 and WT rice seeds --- p.64 / Chapter 4.2.2 --- Pepsin Digestibility of major storage protein fractions in GT3 and WT rice --- p.68 / Chapter 4.2.3 --- Summary of Pepsin Digestibility Assay --- p.74 / Chapter 4.3 --- Mouse Model I --- p.75 / Chapter 4.3.1 --- Protein-specific IgE levels --- p.75 / Chapter 4.3.2 --- Protein-specific IgG1 and IgG2a levels --- p.77 / Chapter 4.3.3 --- Allergic Response Test --- p.79 / Chapter 4.3.4 --- Summary from Mouse Model I --- p.81 / Chapter 4.4 --- Mouse Model II --- p.83 / Chapter 4.4.1 --- Proteins specific IgE levels --- p.84 / Chapter 4.4.2 --- Proteins specific IgG1 and IgG2a levels --- p.85 / Chapter 4.4.3 --- Allergic Response Test --- p.87 / Chapter 4.4.4 --- Summary from Mouse Model II --- p.88 / Chapter 4.5 --- Mouse Model III --- p.90 / Chapter 4.5.1 --- Protein-specific IgE levels --- p.90 / Chapter 4.5.2 --- Proteins specific IgG1 and IgG2a levels --- p.91 / Chapter 4.5.3 --- Allergic Response Test --- p.93 / Chapter 4.5.4 --- Summary from Mouse Model III --- p.93 / Chapter 4.6 --- Potential allergenicity of rice glutelin by bioinformatics and epitope mapping --- p.94 / Chapter 4.6.1 --- Bioinformatic analysis --- p.94 / Chapter 4.6.2 --- ELISA analysis of synthesized epitopes --- p.97 / Chapter 4.6.3 --- Western Blot Analysis --- p.99 / Chapter 4.6.4 --- IgE-binding assay --- p.103 / Chapter Chapter 5. --- Conclusion and Future Perspectives --- p.109 / References --- p.111

by Ng Wing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 156-174). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / List of Graphs --- p.xvii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.3 / Chapter 2.1.1 --- Historical background --- p.3 / Chapter 2.1.2 --- Physiological Roles --- p.5 / Chapter 2.1.3 --- Molecular properties --- p.8 / Chapter 2.1.4 --- Biochemical properties --- p.9 / Chapter 2.1.5 --- Comparison to G-CSF of other species --- p.11 / Chapter 2.1.6 --- Biological Activities --- p.12 / Chapter 2.1.7 --- Clinical Applications --- p.14 / Chapter 2.1.7.1 --- Clinical use in myelosuppressive chemotherapy and neutropenic fever --- p.14 / Chapter 2.1.7.2 --- Clinical use in bone marrow transplantation (BMT) and peripheral blood progenitor cell (PBPC) transplantation --- p.14 / Chapter 2.1.7.3 --- Clinical use in HIV infection --- p.16 / Chapter 2.1.7.4 --- Clinical use in diabetes mellitus --- p.17 / Chapter 2.1.7.5 --- Clinical use in severe chronic neutropenia --- p.18 / Chapter 2.1.7.6 --- Future prospects --- p.18 / Chapter 2.1.7.7 --- Dosages and adverse effects --- p.19 / Chapter 2.1.8 --- Economic value --- p.20 / Chapter 2.2 --- Plant as bioractor --- p.20 / Chapter 2.2.1 --- Medical molecular farming --- p.20 / Chapter 2.2.2 --- Commercial biopharmaceutical proteins --- p.25 / Chapter 2.2.3 --- Transgenic plants producing hematopoietic growth factors --- p.25 / Chapter 2.2.3.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.26 / Chapter 2.2.3.2 --- Interleukin-2 (IL-2) --- p.28 / Chapter 2.3 --- Rice as expression system --- p.29 / Chapter 2.3.1 --- Characteristics --- p.29 / Chapter 2.3.2 --- Advantages of using rice as bioreactor --- p.30 / Chapter 2.3.3 --- Previous studies --- p.31 / Chapter 2.3.4 --- Transformation method --- p.33 / Chapter 2.3.5 --- Super-binary vector --- p.34 / Chapter 2.4 --- Strategies for enhancing protein expression level --- p.36 / Chapter 2.4.1 --- Vacuolar targeting --- p.36 / Chapter 2.4.1.1 --- Protein targeting signals --- p.38 / Chapter 2.4.1.2 --- Binding protein of 80kDa (BP-80) --- p.39 / Chapter 2.4.1.3 --- a-Tonoplast intrinsic protein (α-TIP) --- p.39 / Chapter 2.4.1.4 --- Receptor homology region-transmembrane domain-Ring H2 motif (RMR) --- p.40 / Chapter 2.4.2 --- Fusion with glutelin in rice --- p.41 / Chapter 2.5 --- Hypotheses and aims of this study --- p.43 / Chapter Chapter 3 --- Materials and Methods --- p.45 / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Chemicals --- p.45 / Chapter 3.3 --- Bacterial strains --- p.46 / Chapter 3.4 --- Chimeric genes construction --- p.46 / Chapter 3.4.1 --- Protein targeting constructs --- p.51 / Chapter 3.4.2 --- Enterokinase site constructs --- p.60 / Chapter 3.4.3 --- Glutein signal peptide constructs --- p.65 / Chapter 3.4.4 --- Glutelin fusion constructs --- p.70 / Chapter 3.4.5 --- Sequence fidelity of chimeric genes --- p.77 / Chapter 3.4.6 --- Cloning of chimeric genes into rice super-binary vector --- p.77 / Chapter 3.5 --- Rice transformation --- p.79 / Chapter 3.5.1 --- Plant materials --- p.79 / Chapter 3.5.2 --- Agrobacterium transformation --- p.79 / Chapter 3.5.3 --- A grobacterium-mediated transformation of rice --- p.79 / Chapter 3.6 --- Transgenic expression --- p.81 / Chapter 3.6.1 --- Extraction of leaf genomic DNA --- p.81 / Chapter 3.6.2 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.82 / Chapter 3.6.3 --- Southern blot analysis --- p.83 / Chapter 3.6.4 --- Extraction of total RNA from immature rice seeds --- p.84 / Chapter 3.6.5 --- Northern blot analysis --- p.85 / Chapter 3.6.6 --- Protein extraction --- p.86 / Chapter 3.6.7 --- Tricine SDS-PAGE --- p.86 / Chapter 3.6.8 --- Western blot analysis --- p.87 / Chapter 3.6.9 --- Enterokinase digestion of EK fusion proteins --- p.88 / Chapter 3.7 --- Confocal immunoflorescence studies of rhG-CSF in rice grain --- p.89 / Chapter 3.7.1 --- Preparation of sample sections --- p.89 / Chapter 3.7.2 --- Double-labeling of fluorescence probes --- p.89 / Chapter 3.7.3 --- Image collection --- p.90 / Chapter 3.8 --- Functional analysis of rhG-CSF --- p.91 / Chapter 3.8.1 --- Culture of NFS-60 cells --- p.91 / Chapter 3.8.2 --- MTT cell proliferation assay --- p.92 / Chapter 3.9 --- Bacterial expression of anti-hG-CSF --- p.93 / Chapter 3.9.1 --- pET expression in E. coli --- p.93 / Chapter 3.9.2 --- Purification of His-hG-CSF --- p.97 / Chapter 3.9.3 --- Immunization of rabbits --- p.97 / Chapter Chapter 4 --- Results --- p.99 / Chapter 4.1 --- Construction of chimeric genes for rice transformation --- p.99 / Chapter 4.2 --- "Rice transformation, selection and regeneration" --- p.103 / Chapter 4.3 --- Southern blot analysis --- p.105 / Chapter 4.4 --- Northern blot analysis --- p.109 / Chapter 4.5 --- Western blot analysis --- p.114 / Chapter 4.6 --- Enterokinase digestion of EK fusion proteins --- p.125 / Chapter 4.7 --- Confocal immunofluorescence studies of rhG-CSF in transgenic rice grain --- p.128 / Chapter 4.8 --- Functional analysis of rhG-CSF --- p.132 / Chapter 4.9 --- Bacterial expression of anti-hG-CSF --- p.135 / Chapter 4.9.1 --- Expression and purification of recombinant His-hG-CSF in E. coli --- p.135 / Chapter 4.9.2 --- Titer and specificity of the anti-serum --- p.137 / Chapter Chapter 5 --- Discussion --- p.139 / Chapter 5.1 --- Introduction --- p.139 / Chapter 5.2 --- Fusion of hG-CSF with protein sorting determinants --- p.141 / Chapter 5.3 --- Fusion of hG-CSF with rice glutelin --- p.145 / Chapter 5.4 --- Glutelin signal peptide --- p.146 / Chapter 5.5 --- O-glycosylation --- p.148 / Chapter 5.6 --- Enterokinase digestion --- p.148 / Chapter 5.7 --- Expression level of rhG-CSF --- p.149 / Chapter 5.8 --- Functional analysis of rhG-CSF --- p.151 / Chapter 5.9 --- Future perspectives --- p.151 / Chapter Chapter 6 --- Conclusion --- p.155 / References --- p.156

by Wai Ching Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 154-159). / Abstracts in English and Chinese. / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / List of Abbreviations --- p.viii / Table of contents --- p.x / List of Tables --- p.xiv / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.4 / Chapter 2.1 --- Plant lectins --- p.4 / Chapter 2.1.1 --- Introduction --- p.4 / Chapter 2.1.2 --- Definition and subdivision of plant lectins --- p.4 / Chapter 2.2 --- Monocot mannose-binding lectins --- p.6 / Chapter 2.2.1 --- Occurrence and carbohydrate binding specificity --- p.6 / Chapter 2.2.2 --- Molecular structure and amino acid sequence --- p.7 / Chapter 2.2.3 --- "Molecular cloning, biosynthesis and post-translational modification" --- p.10 / Chapter 2.2.4 --- Mannose-binding lectins of Family Liliaceae --- p.11 / Chapter 2.2.4.1 --- Tulipa gesneriana lectins (TGL) --- p.12 / Chapter 2.2.4.2 --- Aloe arborescens lectins (AAL) --- p.13 / Chapter 2.2.4.3 --- Polygonatum multiflorum agglutinin (PMA) and lectin-related protein --- p.14 / Chapter 2.3 --- Polygonatum odoratum lectins (POL) --- p.15 / Chapter 2.3.1 --- Isolation and purification of POL from Yu Chu --- p.15 / Chapter 2.3.2 --- Agglutinating activity and anti-viral activities of POL --- p.17 / Chapter 2.3.3 --- Bacterial expression of POL in Escherichia coli --- p.18 / Chapter 2.4 --- Plant-based production of recombinant proteins --- p.20 / Chapter 2.4.1 --- Advantages of using plants as expression system --- p.20 / Chapter 2.4.2 --- Plant-derived recombinant proteins --- p.22 / Chapter 2.5 --- Expression of heterologous proteins in rice --- p.24 / Chapter 2.5.1 --- The facts of rice --- p.24 / Chapter 2.5.2 --- Rice storage proteins --- p.25 / Chapter 2.5.2 --- Expression of lysine-rich protein (LRP)/glutelin fusion proteinin rice seeds --- p.28 / Chapter 2.5.3 --- Expression of Galanthus nivalis agglutinin in rice --- p.29 / Chapter 2.6 --- Protein trafficking in plants --- p.30 / Chapter 2.6.1 --- Golgi-dependent pathways --- p.30 / Chapter 2.6.2 --- Golgi-independent pathway --- p.32 / Chapter 2.6.3 --- Expression of protein targeting determinants in tobacco plants and suspension cells --- p.33 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Chemcials --- p.35 / Chapter 3.3 --- Bacterial strains --- p.35 / Chapter 3.4 --- Cloning of POL cDNA --- p.36 / Chapter 3.4.1 --- Plant materials --- p.36 / Chapter 3.4.2 --- RNA extraction --- p.36 / Chapter 3.4.3 --- RT-PCR amplification of POL cDNA --- p.36 / Chapter 3.4.4 --- 5'RACE and 3'RACE --- p.38 / Chapter 3.4.5 --- Sequencing of POL cDNA --- p.39 / Chapter 3.5 --- Analysis of POL protein --- p.40 / Chapter 3.5.1 --- Protein extraction and Tricine-SDS PAGE --- p.40 / Chapter 3.5.2 --- Western blot analysis --- p.41 / Chapter 3.6 --- Chimeric gene construction --- p.42 / Chapter 3.6.1 --- Construction of the Cauliflower mosaic virus (CaMV)35S promoter/POL constructs --- p.44 / Chapter 3.6.2 --- Construction of the glutelin-1 promoter/POL constructs --- p.48 / Chapter 3.6.3 --- Sequence fidelity of chimeric genes --- p.55 / Chapter 3.7 --- Expression of transgenes in rice --- p.55 / Chapter 3.7.1 --- Plant materials --- p.55 / Chapter 3.7.2 --- Agrobacterium transformation --- p.55 / Chapter 3.7.3 --- Callus induction --- p.56 / Chapter 3.7.4 --- Agrobacterium culture and rice transformation --- p.56 / Chapter 3.7.5 --- Selection and regeneration of rice callus --- p.56 / Chapter 3.7.6 --- Isolation of genomic DNA --- p.58 / Chapter 3.7.7 --- Southern blot analysis --- p.58 / Chapter 3.7.8 --- Extraction of leaf total RNA --- p.59 / Chapter 3.7.9 --- Extraction of seed total RNA --- p.59 / Chapter 3.7.10 --- Northern blot analysis --- p.60 / Chapter 3.7.11 --- Protein extraction and Tricine SDS-PAGE --- p.60 / Chapter 3.7.12 --- Western blot analysis --- p.61 / Chapter 3.8 --- Cytopathic effect (CPE) reduction assay --- p.61 / Chapter 3.8.1 --- Protein extraction --- p.61 / Chapter 3.8.2 --- CPE reduction assay --- p.62 / Chapter 3.9 --- Confocal immunofluorescence --- p.63 / Chapter 3.9.1 --- Preparation of sections --- p.63 / Chapter 3.9.2 --- Labelling of fluorescence probes --- p.63 / Chapter 3.9.3 --- Image collection --- p.64 / Chapter Chapter 4 --- Results --- p.65 / Chapter 4.1 --- Cloning of POL cDNA from Yu Chu --- p.65 / Chapter 4.1.1 --- RNA extraction and partial POL cDNA amplification --- p.65 / Chapter 4.1.2 --- 5'RACE and 3'RACE --- p.67 / Chapter 4.1.3 --- Sequencing of POL cDNA --- p.68 / Chapter 4.1.4 --- Sequences comparison of POL and Liliaceae lectins --- p.75 / Chapter 4.2 --- Occurence of POL protein in Yu Chu plant --- p.77 / Chapter 4.3 --- Constitutional expression of POL in rice --- p.79 / Chapter 4.3.1 --- Construction of Cauliflower mosaic virus 35S promoter constructs --- p.80 / Chapter 4.3.2 --- Southern blot analysis --- p.82 / Chapter 4.3.3 --- Northern blot analysis --- p.84 / Chapter 4.3.4 --- Western blot analysis --- p.85 / Chapter 4.3.5 --- Western blot analysis of 35S/POL T1 plant --- p.87 / Chapter 4.4 --- Seed-specific expression of POL in rice --- p.88 / Chapter 4.4.1 --- Construction of the glutelin-1 promoter constructs --- p.89 / Chapter 4.4.2 --- Southern blot analysis --- p.92 / Chapter 4.4.3 --- Northern blot analysis --- p.96 / Chapter 4.4.4 --- Western blot analysis --- p.101 / Chapter 4.4.5 --- Western blot analysis of POL-BP-8O and POL-α-TIP T1 transgenic plants --- p.117 / Chapter 4.5 --- Cytopathic effect (CPE) reduction assay --- p.122 / Chapter 4.6 --- Confocal immunofluorescence studies --- p.125 / Chapter Chapter 5 --- Discussion --- p.134 / Chapter 5.1 --- Cloning of POL cDNA --- p.134 / Chapter 5.2 --- Analysis of constitutional expression of POL in rice --- p.136 / Chapter 5.3 --- Analysis of seed-specific expression of POL in rice --- p.138 / Chapter 5.4 --- Localization of POL in POL-BP-8O and POL-α-TIP transgenic rice seeds --- p.146 / Chapter 5.5 --- Cytopathic effect (CPE) reduction assay --- p.148 / Chapter 5.6 --- Future prospects --- p.151 / Chapter Chapter 6 --- Conclusion --- p.153