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Characterization of lysine-rich protein (LRP) in winged bean.

Wong Ho Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.III / Abstract --- p.IV / 摘要 --- p.VI / List of Tables --- p.VIII / List of Figures --- p.IX / List of Abbreviations --- p.XI / Table of Contents --- p.XIII / Chapter 1 --- General introduction --- p.1 / Chapter 2 --- Literature reviews --- p.4 / Chapter 2.1 --- LRP and winged bean --- p.4 / Chapter 2.1.1 --- Nutritional values of crop plants --- p.4 / Chapter 2.1.2 --- Lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.1 --- Identification of lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.2 --- Cloning cDNA for WBLRP --- p.7 / Chapter 2.1.2.3 --- Transgenic Expression of LRP in other plants --- p.8 / Chapter 2.1.3 --- Unknowns remained --- p.8 / Chapter 2.2 --- Food allergy and gastro-immunity --- p.10 / Chapter 2.2.1 --- What is allergy? 一 A brief introduction --- p.10 / Chapter 2.2.2 --- Food allergy and its symptoms --- p.12 / Chapter 2.2.3 --- Gastrointestinal immunity --- p.13 / Chapter 2.2.4 --- Possible mechanism of food allergy --- p.16 / Chapter 2.2.5 --- Available tests and limitations --- p.18 / Chapter 2.2.6 --- Radioallergosorbent test (RAST) --- p.19 / Chapter 2.2.7 --- Digestibility test --- p.20 / Chapter 2.2.8 --- Betv-1 Allergen Family --- p.21 / Proteins --- p.23 / Chapter 2.3 --- Pathogenesis-related proteins --- p.23 / Chapter 2.3.1 --- Defense-related proteins and pathogenesis-related proteins (PRs) --- p.23 / Chapter 2.3.2 --- Class 10 PR proteins (PR-10s) --- p.25 / Chapter 2.3.3 --- The expression patterns of PR-10s --- p.27 / Chapter 2.3.3.1 --- Pathogens-induced and signal-induced expression --- p.27 / Chapter 2.3.3.2 --- Spatially- and developmentally-regulated expression --- p.28 / Chapter 2.3.3.3 --- Other induction patterns --- p.29 / Chapter 2.3.4 --- Functions ofPR-10s --- p.30 / Chapter 2.4 --- Development of hypotheses and experiments --- p.32 / Chapter 3 --- Materials and methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials --- p.38 / Chapter 3.2.1 --- Chemicals --- p.38 / Chapter 3.2.2 --- Apparatus and commercial kits --- p.39 / Chapter 3.2.3 --- Vectors and bacterial strains --- p.39 / Chapter 3.2.4 --- Plant and animal materials --- p.40 / Chapter 3.2.5 --- Computer software --- p.40 / Chapter 3.3 --- Purification of LRP --- p.41 / Chapter 3.3.1 --- Purification of LRP from winged bean --- p.41 / Chapter 3.3.1.1 --- Extraction of total protein --- p.41 / Chapter 3.3.1.2 --- Differential pI precipitation --- p.41 / Chapter 3.3.1.3 --- Determination of the pI point of LRP --- p.42 / Chapter 3.3.1.4 --- Native tricine-PAGE and gel elution --- p.42 / Chapter 3.3.2 --- Purification from E. coli --- p.45 / Chapter 3.3.2.1 --- Construction of pET vector expressing recombinant LRP (rLRP) --- p.45 / Chapter 3.3.2.2 --- Expression of rLRP --- p.50 / Chapter 3.3.2.3 --- Purification by gel electrophoresis and gel band elution --- p.50 / Chapter 3.4 --- Anti-serum production --- p.52 / Chapter 3.5 --- Allergy tests --- p.53 / Chapter 3.5.1 --- Pepsin digestion --- p.53 / Chapter 3.5.1.1 --- Determination of optimal concentration of pepsin --- p.53 / Chapter 3.5.1.2 --- Pepsin digestion of allergenic and non-allergenic model proteins --- p.55 / Chapter 3.5.1.3 --- Pepsin digestion of LRP and immunodetection --- p.55 / Chapter 3.5.2 --- Trypsin digestion --- p.56 / Chapter 3.5.2.1 --- Determination of optimal trypsin concentration --- p.56 / Chapter 3.5.2.2 --- Trypsin digestion of allergenic and non-allergenic model proteins --- p.57 / Chapter 3.5.2.3 --- Trypsin digestion of LRP and immuno-detection --- p.57 / Chapter 3.5.3 --- Pepsin and trypsin digestion --- p.58 / Chapter 3.5.3.1 --- Digestions of allergenic model proteins --- p.58 / Chapter 3.5.3.2 --- Digestion of LRP --- p.58 / Chapter 3.5.4 --- IgE binding tests --- p.58 / Chapter 3.6 --- Physiology studies --- p.59 / Chapter 3.6.1 --- Preparation for the studies --- p.59 / Chapter 3.6.1.1 --- Growing winged bean in the field --- p.59 / Chapter 3.6.1.2 --- Growing winged bean in sterile conditions --- p.60 / Chapter 3.6.1.3 --- Production ofLRP-cDNA probe --- p.60 / Chapter 3.6.2 --- Detecting the expression of LRP in winged bean --- p.61 / Chapter 3.6.2.1 --- RNA extraction --- p.61 / Chapter 3.6.2.2 --- RT-PCR and DNA sequencing --- p.62 / Chapter 3.6.2.3 --- RNA electrophoresis and northern blot analysis --- p.63 / Chapter 3.6.2.4 --- Protein extraction --- p.63 / Chapter 3.6.2.5 --- Western blot and immuno-detection --- p.63 / Chapter 3.6.3 --- Expression of LRP in germinating winged bean seeds --- p.64 / Chapter 3.6.3.1 --- Seed germination --- p.64 / Chapter 3.6.3.2 --- Detection of LRP in germinating seeds --- p.64 / Chapter 3.6.4 --- RNase activity test --- p.65 / Chapter 4 --- Results --- p.67 / Chapter 4.1 --- Purification of LRP --- p.67 / Chapter 4.1.1 --- Purification from winged bean --- p.67 / Chapter 4.1.1.1 --- Identification of pI point of LRP --- p.67 / Chapter 4.1.1.2 --- Native tricine PAGE and gel elution --- p.70 / Chapter 4.1.2 --- Purification from E. coli --- p.71 / Chapter 4.1.2.1 --- Construction of pET-LRP vector --- p.71 / Chapter 4.1.2.2 --- Expression of rLRP and gel purification --- p.74 / Chapter 4.2 --- Antiserum production --- p.76 / Chapter 4.3 --- Allergy tests --- p.81 / Chapter 4.3.1 --- Pepsin digestion --- p.81 / Chapter 4.3.2 --- Trypsin digestion --- p.89 / Chapter 4.3.3 --- Pepsin and trypsin digestion --- p.96 / Chapter 4.3.4 --- Human serum IgE binding test --- p.104 / Chapter 4.4 --- Physiological studies --- p.105 / Chapter 4.4.1 --- Samples preparation --- p.105 / Chapter 4.4.2 --- RT-PCR and DNA sequencing --- p.105 / Chapter 4.4.3 --- Expression profile of WBLRP in winged bean somatic organs --- p.108 / Chapter 4.4.4 --- Expression profile ofWBLRP in winged bean flower --- p.111 / Chapter 4.4.5 --- Expression profile ofWBLRP in winged bean maturing seeds --- p.114 / Chapter 4.4.6 --- Expression profile of WBLRP gene in winged bean germinating seeds --- p.117 / Chapter 4.4.7 --- Functional assay of LRP --- p.121 / Chapter 5 --- Discussion --- p.124 / Chapter 5.1 --- LRP purification and antibody production --- p.124 / Chapter 5.2 --- Allergy tests --- p.125 / Chapter 5.3 --- Expression of LRP in WB --- p.131 / Chapter 5.4 --- Functional assay of LRP --- p.134 / Chapter 5.5 --- Hypothesis Testing --- p.135 / Chapter 5.6 --- Future prospective, --- p.136 / Chapter 6 --- Conclusion --- p.138 / Chapter 7 --- References --- p.140

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324232
Date January 2003
ContributorsWong, Ho Wan., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvi, 153 leaves : ill. (some col.) ; 30 cm.
RightsUse 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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