Lam Sze Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-200). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Table of contents --- p.ii / Abstract --- p.xii / 撮要 --- p.xv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / TABLE OF CONTENTS / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Overview of Chitinases --- p.3 / Chapter 1.1.1. --- Classification of Chitinases --- p.7 / Chapter 1.1.1.1. --- Family 19 Chitinases --- p.7 / Chapter 1.1.1.1.1. --- Class I Chitinases --- p.9 / Chapter 1.1.1.1.2. --- Class II Chitinases --- p.10 / Chapter 1.1.1.1.3. --- Class IV Chitinases --- p.10 / Chapter 1.1.1.1.4. --- Class V Chitinases --- p.11 / Chapter 1.1.1.1.5. --- Class VI Chitinases --- p.11 / Chapter 1.1.1.2. --- Family 18 Chitinases --- p.12 / Chapter 1.1.1.2.1. --- PR-8/Class III Chitinases --- p.12 / Chapter 1.1.1.2.2. --- PR-11 Chitinases --- p.15 / Chapter 1.1.1.3. --- The PR-4 Family --- p.16 / Chapter 1.1.2. --- Catalytic Mechanism of Chitinases --- p.19 / Chapter 1.1.2.1. --- Catalytic Mechanism of Family 18 Chitinases --- p.20 / Chapter 1.1.2.2. --- Catalytic Mechanism of Family 19 Chitinases --- p.21 / Chapter 1.1.3. --- Biological Properties of Chitinases --- p.22 / Chapter 1.1.3.1. --- Antifungal Activity of Chitinases in vitro --- p.22 / Chapter 1.1.3.2. --- Antifungal Activity of Chitinases in vivo --- p.23 / Chapter 1.1.3.3. --- Other Functions --- p.23 / Chapter 1.2. --- Overview of Ribonucleases --- p.25 / Chapter 1.2.1. --- Classification of Ribonucleases --- p.26 / Chapter 1.2.1.1. --- RNase T1 Family --- p.26 / Chapter 1.2.1.1.1. --- Action Mechanism of RNase T1 Family --- p.32 / Chapter 1.2.1.2. --- RNase T2 Family --- p.34 / Chapter 1.2.1.2.1. --- Action Mechanism of RNase T2 Family --- p.36 / Chapter 1.2.2. --- Biological Activities of Plant Ribonucleases --- p.38 / Chapter 1.2.2.1. --- Phosphate Remobilization --- p.38 / Chapter 1.2.2.2. --- Senescence --- p.39 / Chapter 1.2.2.3. --- Programmed Cell Death --- p.40 / Chapter 1.2.2.4. --- Plant Defense --- p.41 / Chapter 1.2.2.5. --- RNA Processing and Decay --- p.43 / Chapter 1.2.2.6. --- Antitumor Activities --- p.43 / Chapter 1.3. --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.45 / Chapter 1.3.1. --- General properties of RIPs --- p.46 / Chapter 1.3.1.1. --- Classification of RIPs --- p.46 / Chapter 1.3.2. --- Activities of Ribosome-inactivating Proteins --- p.52 / Chapter 1.3.2.1. --- RNA N-glycosidase activity --- p.52 / Chapter 1.3.2.2. --- Protein synthesis inhibitory activity --- p.58 / Chapter 1.3.2.3. --- Abortifacient activity --- p.59 / Chapter 1.3.2.4. --- Immunosuppressive activity --- p.60 / Chapter 1.3.2.5. --- Antiviral activity --- p.61 / Chapter 1.3.3. --- Roles of RIPs in plants --- p.63 / Chapter 1.3.3.1. --- Defensive role of RIPs in plants --- p.63 / Chapter 1.3.3.2. --- Role of RIPs in stress adaptation in plants --- p.66 / Chapter 1.3.4. --- Possible application of RIPs --- p.67 / Chapter 1.3.4.1. --- Use of RIPs in therapies --- p.67 / Chapter 1.3.4.1.1. --- Antiviral agents --- p.67 / Chapter 1.3.4.1.2. --- Immunotoxins --- p.68 / Chapter 1.3.4.1.3. --- Anti-HIV drugs --- p.69 / Chapter 1.3.4.2. --- Use of RIPs in agriculture --- p.71 / Chapter 1.4. --- Overview of the PR-5 Family: Thaumatin-Like Proteins (TLPs) --- p.72 / Chapter 1.4.1. --- Occurrence of Thaumatin-Like Proteins --- p.76 / Chapter 1.4.2. --- Biological properties of TLPs --- p.77 / Chapter 1.4.2.1. --- Antifungal Activity --- p.77 / Chapter 1.4.2.2. --- TLPs as Anti-Freeze Protein --- p.78 / Chapter 1.4.3. --- Biotechnological Application ´ؤ Transgenic Plants --- p.79 / Chapter Chapter 2 --- Materials and Methods --- p.81 / Chapter 2.1. --- Materials --- p.81 / Chapter 2.2. --- Preparation of Crude Extract --- p.82 / Chapter 2.3. --- Purification --- p.83 / Chapter 2.4. --- Chromatography --- p.84 / Chapter 2.4.1. --- CM-Cellulose Chromatography --- p.84 / Chapter 2.4.2. --- Mono S® HR 5/5 and Mono Q® HR 5/5 --- p.85 / Chapter 2.4.3. --- Affi-gel Blue gel --- p.86 / Chapter 2.4.4. --- Superdex75 --- p.87 / Chapter 2.5. --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 2.6. --- Protein Concentration Determination --- p.89 / Chapter 2.7. --- Preparation of Rabbit Reticulocyte Lysate --- p.90 / Chapter 2.8. --- Determination of N-terminal Amino Acid Sequence --- p.91 / Chapter 2.9. --- Biological Activity Assays --- p.92 / Chapter 2.9.1. --- Assay for Antifungal Activity --- p.92 / Chapter 2.9.2. --- Assay for Cell-Free Translation Inhibitory Activity --- p.93 / Chapter 2.9.3. --- Assay of Cytotoxic Activity on Cancer Cell Lines --- p.94 / Chapter 2.9.4. --- Assay for HIV-1 Reverse Transcriptase (RT) Inhibitory Activity --- p.95 / Chapter 2.9.5. --- Assay of Mitogenic Activity --- p.97 / Chapter 2.9.6. --- Assay for N-Glycosidase Activity --- p.98 / Chapter 2.9.6.1. --- RNA Extraction --- p.98 / Chapter 2.9.6.2. --- Aniline Treatment --- p.99 / Chapter 2.9.6.3. --- Formaldehyde Gel Electrophoresis --- p.99 / Chapter 2.9.7. --- Assay of Ribonuclease Activity --- p.100 / Chapter 2.9.7.1. --- Assay for Yeast tRNA --- p.100 / Chapter 2.9.7.2. --- Activity toward Polyhomoribonucleotides --- p.100 / Chapter Chapter 3 --- Purification and Characterization of Pathogenesis-Related Proteins from their Respective Sources --- p.101 / Chapter 3.1. --- Purification and Characterization of Chitinase and Ribonuclease from the Roots of Panax notoginseng --- p.102 / Chapter 3.1.1. --- Introduction --- p.102 / Chapter 3.1.2. --- Results --- p.104 / Chapter 3.1.3. --- Purification --- p.107 / Chapter 3.1.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.108 / Chapter 3.1.3.2. --- Affinity Chromatography on Affi-gel Blue gel --- p.111 / Chapter 3.1.3.3. --- Cation-Exchange Chromatography on Mono S Column --- p.114 / Chapter 3.1.3.4. --- Gel Filtration on Superdex 75 Column --- p.115 / Chapter 3.1.4. --- Characterization of Chitinase --- p.117 / Chapter 3.1.4.1. --- N-terminal Amino Acid Sequence --- p.117 / Chapter 3.1.4.2. --- Assay for Antifungal Activity --- p.118 / Chapter 3.1.4.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.120 / Chapter 3.1.4.4. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.120 / Chapter 3.1.5. --- Characterization of Ribonuclease --- p.121 / Chapter 3.1.5.1. --- N-terminal Amino Acid Sequence --- p.121 / Chapter 3.1.5.2. --- Assay for Ribonuclease Activity --- p.122 / Chapter 3.1.5.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.125 / Chapter 3.1.5.4. --- Assay for Antifungal Activity --- p.125 / Chapter 3.1.5.5. --- Assay for Antiproliferative Activity --- p.126 / Chapter 3.1.6. --- Discussion --- p.127 / Chapter 3.2. --- Purification and Characterization of Ribosome-Inactivating Protein and Antifungal Protein from the mushroom Lyophyllum shimeji --- p.131 / Chapter 3.2.1. --- Introduction --- p.131 / Chapter 3.2.2. --- Results --- p.132 / Chapter 3.2.3. --- Purification --- p.134 / Chapter 3.2.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.135 / Chapter 3.2.3.2. --- Affinity Chromatography on Affi-gel Blue Gel --- p.137 / Chapter 3.2.3.3. --- Cation-Exchange Chromatography on Mono S --- p.140 / Chapter 3.2.4. --- Characterization of Ribosome-Inactivating Protein and Antifungal Protein from Lyophyllum shimeji --- p.142 / Chapter 3.2.4.1. --- N-terminal Amino Acid Sequence --- p.142 / Chapter 3.2.4.2. --- Assay for Antifungal Activity --- p.144 / Chapter 3.2.4.3. --- Assay for N-glycosidase Activity --- p.147 / Chapter 3.2.4.4. --- Assay for Mitogenic Activity --- p.147 / Chapter 3.2.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.148 / Chapter 3.2.5. --- Discussion --- p.150 / Chapter 3.3. --- Purification and Characterization of Ribosome-inactivating Protein from the Hypsizigus marmoreus --- p.153 / Chapter 3.3.1. --- Introduction --- p.153 / Chapter 3.3.2. --- Result --- p.154 / Chapter 3.3.3. --- Purification --- p.155 / Chapter 3.3.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.156 / Chapter 3.3.3.2. --- Affinity-Chromatography on Affi-gel Blue Gel --- p.158 / Chapter 3.3.3.3. --- Anion-Exchange Chromatography on Mono Q Column --- p.160 / Chapter 3.3.4. --- Characterization of Ribosome-inactivating Protein from Hypsizigus marmoreus --- p.162 / Chapter 3.3.4.1. --- N-terminal Amino Acid Sequence --- p.162 / Chapter 3.3.4.2. --- Assay for Cell-Free Translation-Inhibiting Activity --- p.163 / Chapter 3.3.4.3. --- Assay for Antifungal Activity --- p.164 / Chapter 3.3.4.4. --- Assay for N-glycosidase Activity --- p.166 / Chapter 3.3.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.166 / Chapter 3.3.4.6. --- Assay for mitogenic Activity --- p.167 / Chapter 3.3.4.7. --- Assay for Antiproliferative Activity --- p.167 / Chapter 3.3.5. --- Discussion --- p.159 / Chapter Chapter 4 --- General Discussion --- p.170 / References --- p.172

植物合成多種發病機理相關蛋白以對抗病原體的侵襲。植物發病機理相關蛋白包括：核糖核酸酶；抗真菌蛋白；凝集素；胰蛋白酶抑制因子等。這些發病機理相關蛋白具有抗病毒，抗細菌，抗真菌，免疫調節及抗腫瘤等活性。從六種植物中提純了七個發病機理相關蛋白，包括三個凝集素，一個核糖核酸酶，兩個種抗真菌蛋白及一個胰蛋白酶抑制因子。 / 從西洋參須中提純了新的核糖核酸酶。核糖核酸酶分子量為26kDa，具有特异N末端氨基酸序列。此核糖核酸酶在 pH5 及 60℃ 條件下活性最高。它能抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 從粉色菜豆及日本大花豆中提純了兩種凝集素。它們由兩個分子量為32kDa的亞基構成雙倍體。他們的活性穩定于0-60℃及3-12 pH。粉色菜豆凝集素的特异性糖基為木糖，日本大花豆凝集素的特异性糖基為半乳糖。從太子參中提純的凝集素分子量為33kDa，其活性穩定于0-60℃及2-5 pH。 這三種凝集素都具有抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 提純的胰蛋白酶抑制因子分子量為21kDa。具有高耐熱及耐酸鹼性并表現出抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。從豇豆中提純的抗真菌肽分子量為5447Da，具有類防御素N末端氨基酸序列。 / Plants produce a diversity of proteins with antipathogenic activities. These proteins comprise among others, (i) ribonucleases, (ii) antifungal proteins, (iii) lectins and (iv) trypsin inhibitor with antiviral, antifungal and anticancer activities. The aim of this project was to isolate antipathogenic plant proteins including a ribonuclease from American ginseng branch roots, a trypsin inhibitor from rambutan seeds, defensin-like antifungal peptides from borlotti beans and king pole beans, and lectins from borlotti beans, Japanese large pinto beans and Pseudostellaria heterophylla. / The isolated 26-kDa ginseng branch root ribonuclease was monomeric with a novel N-terminal amino acid sequence. It exhibited maximal robonucleolytic activity toward yeast tRNA at pH 5 and 60℃. It inhibited proliferation of MCF7 human breast cancer cells and HepG2 human hepatoma cells. It also inhibited the activity of HIV-1 reverse transcriptase. / Both borlotti bean lectin and Japanese large pinto bean lectin were dimeric with a subunit molecular mass of 32-kDa. They were stable from 0℃ to 60℃ and from pH 3 to pH 12. Borlotti bean lectin was xylose-specific whereas Japanse large pinto bean lectin was galactose-specific. The 33-kDa Pseudostellaria heterophylla lectin could not be inhibited by the simple sugars tested. It was stable from 0℃ to 60℃ and from pH 2 to 5. All three isolated lectins suppressed proliferation of MCF7 and HepG2 cells and inhibited HIV-1 reverse transcriptase. / The isolated 21-kDa rambutan trypsin inhibitor has relatively high pH stability and thermostability, and exhibited HIV-1 reverse transcriptase inhibitory activity and antiproliferative activity toward a variety of tumor cells. The isolated 5447-Da king pole bean defensin-like peptide inhibited fungal growth. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xii / Chapter Chapter 1 --- Overview of Plant Defense-related Protein --- p.1 / Chapter 1.1 --- Overview of Lectins and hemagglutinins --- p.4 / Chapter 1.1.1 --- History and definition of lectins and hemagglutinins --- p.4 / Chapter 1.1.2 --- Occurrence and distribution of plant lectins --- p.6 / Chapter 1.1.3 --- Classification of lectins --- p.7 / Chapter 1.1.3.1 --- Classification of lectins on the basis of overall structure of lectin subunits --- p.7 / Chapter 1.1.3.2 --- Classification of lectins based on binding specificty to carbohydrates --- p.11 / Chapter 1.1.3.3 --- Classification of lectins according to families --- p.12 / Chapter 1.1.3.3.1 --- Legume lectins --- p.12 / Chapter 1.1.3.3.2 --- Monocot mannose-binding lectins --- p.13 / Chapter 1.1.3.3.3 --- Other lectins --- p.14 / Chapter 1.1.4 --- Defensive role of plant lectins --- p.15 / Chapter 1.1.5 --- Applications of plant lectins --- p.18 / Chapter 1.1.5.1 --- The antibacterial activity --- p.18 / Chapter 1.1.5.2 --- Anti-insect activity --- p.19 / Chapter 1.1.5.3 --- Antifungal activity --- p.21 / Chapter 1.1.5.4 --- The antiviral activity --- p.22 / Chapter 1.1.5.5 --- Lectin affinity chromatography --- p.23 / Chapter 1.1.5.6 --- Lectin microarray --- p.23 / Chapter 1.2 --- Overview of Ribonucleases --- p.26 / Chapter 1.2.1 --- History and definition of Ribonucleases --- p.26 / Chapter 1.2.2 --- Classification of Ribonucleases --- p.27 / Chapter 1.2.2.1 --- T1 Ribonucleases family --- p.27 / Chapter 1.2.2.2 --- RNase T2 family --- p.28 / Chapter 1.2.3 --- Biological activities of plant ribonucleases --- p.28 / Chapter 1.2.3.1 --- Phosphate remobilization --- p.28 / Chapter 1.2.3.2 --- Senescence --- p.29 / Chapter 1.2.3.3 --- Programmed cell death --- p.30 / Chapter 1.2.3.4 --- Plant defense --- p.31 / Chapter 1.2.3.5 --- RNA processing and decay --- p.32 / Chapter 1.2.3.6 --- Antitumor activities --- p.33 / Chapter 1.3 --- Other plant pathogen-related proteins --- p.34 / Chapter 1.3.1 --- Overview of chitinase --- p.34 / Chapter 1.3.1.1 --- Classification of chitinases --- p.35 / Chapter 1.3.1.2 --- Biological properties of chitinases --- p.38 / Chapter 1.3.2 --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.41 / Chapter 1.3.2.1 --- Classification of RIPs --- p.42 / Chapter 1.3.2.2 --- Roles of RIPs in plants --- p.44 / Chapter 1.3.2.3 --- Possible application of RIPs --- p.46 / Chapter 1.3.3 --- Overview of thaumatin-like proteins (TLPs) --- p.50 / Chapter 1.3.3.1 --- Occurrence of TLPs --- p.51 / Chapter 1.3.3.2 --- Biological properties of TLPs --- p.52 / Chapter 1.4 --- Aim of this study --- p.54 / Chapter Chapter 2 --- Isolation of a lectin and an antifungal protein from Phaseolus vulgaris cv. Borlotti beans / Chapter 2.1 --- Introduction --- p.55 / Chapter 2.2 --- Materials and Methods --- p.55 / Chapter 2.3 --- Results --- p.64 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Isolation of a lectin from Pinto beans (Phaseolus vulgaris pinto bean) / Chapter 3.1 --- Introduction --- p.82 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.3 --- Results --- p.87 / Chapter 3.4 --- Discussion --- p.103 / Chapter Chapter 4 --- Isolation of a lectin from Pseudostellaria hetorophylla roots / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Materials and Methods --- p.107 / Chapter 4.3 --- Results --- p.110 / Chapter 4.4 --- Discussion --- p.122 / Chapter Chapter 5 --- Isolation of a ribonuclease from branch roots of American ginseng (Panax quinquefolium) / Chapter 5.1 --- Introduction --- p.124 / Chapter 5.2 --- Materials and Methods --- p.126 / Chapter 5.3 --- Results --- p.129 / Chapter 5.4 --- Discussion --- p.142 / Chapter Chapter 6 --- Isolation of a trypsin inhibitor in rambutan (Nephelium lappaceum L) seeds / Chapter 6.1 --- Introduction --- p.144 / Chapter 6.2 --- Materials and Methods --- p.147 / Chapter 6.3 --- Results --- p.152 / Chapter 6.4 --- Discussion --- p.163 / Chapter Chapter 7 --- Isoation of a defensin-like antifungal peptide from Phaseolus vulgaris cv. 'King Pole Bean' / Chapter 7.1 --- Introduction --- p.168 / Chapter 7.2 --- Materials and Methods --- p.170 / Chapter 7.3 --- Results --- p.173 / Chapter 7.4 --- Discussion --- p.181 / Chapter Chapter 8 --- Overall discussion --- p.183 / References --- p.186

Wang Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 124-135). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Table of contents / List of figures / List of tables / List of abbreviations / CHAPTER / Chapter 1. --- Introduction / Chapter 1.1 --- Background of SARS and epidemiology / Chapter 1.2 --- SARS symptoms and infected regions / Chapter 1.3 --- SARS virus / Chapter 1.4 --- Treatment for SARS at present / Chapter 1.5 --- Vaccine development is a more effective way to fight against SARS / Chapter 1.6 --- Vaccine candidates / Chapter 1.6.1 --- Truncated S protein as a vaccine candidate / Chapter 1.6.2 --- Full-length N protein as a vaccine candidate / Chapter 1.7 --- E.coli expression system / Chapter 1.8 --- Baculovirus expression system / Chapter 1.8.1 --- Characteristics of baculovirus / Chapter 1.8.2 --- Infection cycle of baculovirus / Chapter 1.8.3 --- Control of viral gene expression in virus-infected cells / Chapter 1.8.4 --- Merits of baculovirus expression system / Chapter 1.9 --- Aim of study / Chapter 2. --- "Bacterial expression and purification of rS1-1000(E), rS401-1000(E) and rN(E)" / Chapter 2.1 --- Introduction / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Reagents for bacterial culture / Chapter 2.2.2 --- Reagents for agarose gel electrophoresis / Chapter 2.2.3 --- 2'-deoxyribonucleoside 5'-triphosphate (dNTP) mix for polymerase chain reaction (PCR) / Chapter 2.2.4 --- Sonication buffer / Chapter 2.2.5 --- Reagents for immobilized metal affinity chromatography (IMAC) purification / Chapter 2.2.6 --- Reagents for gel filtration chromatography / Chapter 2.2.7 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) / Chapter 2.2.8 --- Reagents for Western blotting / Chapter 2.3 --- Methods / Chapter 2.3.1 --- General techniques in molecular cloning / Chapter 2.3.2 --- "PCR amplification of the S1-400,S401-1000" / Chapter 2.3.3 --- Construction of clone pET-S 1-400 and PET-s401-1000 / Chapter 2.3.4 --- Construction of clone pAC-N / Chapter 2.3.5 --- Expression / Chapter 2.3.6 --- Inclusion bodies preparation / Chapter 2.3.7 --- Inclusion bodies solubilization using urea / Chapter 2.3.8 --- Protein refolding by rapid dilution and dialysis / Chapter 2.3.9 --- Purification of recombinant protein by nickel ion chelating Sepharose fast flow column (IMAC) / Chapter 2.3.10 --- Gel filtration chromatography for further purification / Chapter 2.3.11 --- Bradford assay for the protein concentration analysis / Chapter 2.3.12 --- Protein analysis / Chapter 2.4 --- Results / Chapter 2.4.1 --- SDS-PAGE analysis of the expressed proteins / Chapter 2.4.2 --- Western blot analysis of the bacterial cell lysate / Chapter 2.4.3 --- Protein purification by IMAC / Chapter 2.4.4 --- Purification of rS401-1000(E) by gel filtration / Chapter 2.4.5 --- Determination of production yield of recombinant fusion proteins / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- Expression vector selected for rS1-400(E) and rS401-1000(E) expression / Chapter 2.5.2 --- Protein expression in E.coli / Chapter 2.5.3 --- Purification process / Chapter 3. --- Baculovirus expression and purification of rS401-1000(ACN) and rN(BMN) protein / Chapter 3.1 --- Introduction / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Reagents for insect cell culture and virus work / Chapter 3.3 --- Methods / Chapter 3.3.1 --- "PCR amplification of N and cloning of S401-1000, N genes into the transfer vector pVL1393" / Chapter 3.3.2 --- Cloning of S401-1000 into transfer vector pFastBac HT B / Chapter 3.3.3 --- Virus works / Chapter 3.3.4 --- Identification of recombinant BmNPV or AcMNPV / Chapter 3.3.5 --- Manipulation of silkworm / Chapter 3.3.6 --- Mouse immunization for polyclonal antibody against rN(E) protein / Chapter 3.4 --- Results / Chapter 3.4.1 --- Expression of rN(BMN) in baculovirus / Chapter 3.4.2 --- Expression of rS401-1000(BMN) and rS401-1000(ACN) in baculovirus / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- The expression level of rN(BMN) in both in vitro and invivo / Chapter 3.5.2 --- The rS401-1000(ACN) protein expression level in vitro / Chapter 3.5.3 --- Failure in generating rS401-1000(BMN) / Chapter 3.5.4 --- Purification process of rN(BMN) by IMAC / Chapter 4. --- "Characterization of recombinant rS1-400(E), rN(E), rN(BMN), rS401_1000(E) and rS401-1000(ACN)" / Chapter 4.1 --- Introduction / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Reagents for enzyme-linked immunosorbent assay (ELISA) / Chapter 4.2.2 --- Reagents for purification of human IgG / Chapter 4.2.3 --- Source and identity of Immune sera / Chapter 4.3 --- Methods / Chapter 4.3.1 --- ELISA / Chapter 4.3.2 --- Purification process of human IgG / Chapter 4.4 --- Results / Chapter 4.4.1 --- Validation of Immune sera using SARS viral lysate / Chapter 4.4.2 --- Immunoreactivities of rS1-400(E) and rN(E) against pooled patients sera and normal human serum / Chapter 4.4.3 --- Immunoreactivity comparison of rN(E) and rN(BMN) / Chapter 4.4.4 --- Comparison of the immunoreactivities of rS401-1000(E) and rS401-1000(ACN) / Chapter 4.4.5 --- Immunoreactivity of SARS related proteins against Anti-SARS Antibody (Equine) / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Comparison of the immunoreactivities of SARS related proteins expressed in the present study / References

Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144