Leung Kwan-chi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 215-223). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.iii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Brief description of Momordica charantia --- p.2 / Chapter 1.2 --- Toxicity of RIPs and their potential uses in the treatment of AIDS --- p.3 / Chapter 1.3 --- General mechanism of action of RIPs --- p.6 / Chapter 1.4 --- Structure of αMMC --- p.7 / Chapter 1.5 --- "Antigenicities of αMMC, BMMC and TCS" --- p.13 / Chapter 1.6 --- "Immunosuppressive properties of the abortifacient proteins αMMC, BMMC and TCS" --- p.14 / Chapter 1.7 --- Objectives of our study --- p.15 / Chapter CHAPTER 2 --- EXPRESSION OF FULL-LENGTH αMMC cDNA --- p.20 / Chapter 2.1 --- Expression of αMMC cDNA as a fusion protein --- p.22 / Chapter 2.1.1 --- Materials and methods --- p.22 / Chapter 2.1.1.1 --- Construction of fusion vector pRIT2T/MMC --- p.22 / Chapter 2.1.1.2 --- Preparation of αMMC insert by PCR --- p.26 / Chapter 2.1.1.3 --- Cloning of αMMC cDNA into fusion vector pRIT2T --- p.27 / Chapter 2.1.1.4 --- Transformation --- p.28 / Chapter 2.1.1.5 --- DNA sequencing --- p.29 / Chapter 2.1.1.6 --- Expression of protein A-αMMC fusion cDNA --- p.30 / Chapter 2.1.1.7 --- Preparation of fusion αMMC for affinity chromatography --- p.31 / Chapter 2.1.1.8 --- Affinity chromatography of Protein A-αMMC fusion protein --- p.31 / Chapter 2.1.1.9 --- Cleavage of protein A-αMMC fusion protein by factor Xa --- p.32 / Chapter 2.1.1.10 --- SDS-PAGE analysis --- p.33 / Chapter 2.1.1.11 --- Western blot analysis --- p.33 / Chapter 2.1.1.12 --- Assay of biological activity --- p.35 / Chapter 2.1.2 --- Results --- p.37 / Chapter 2.1.2.1 --- Construction of pRIT2T/MMC --- p.37 / Chapter 2.1.2.2 --- DNA sequencing --- p.40 / Chapter 2.1.2.3 --- Expression of protein A-αMMC fusion cDNA --- p.42 / Chapter 2.1.2.4 --- Purification of protein A-αMMC fusion protein --- p.45 / Chapter 2.1.2.5 --- Cleavage of protein A-aMMC fusion protein --- p.49 / Chapter 2.1.2.6 --- Assay of biological activity --- p.49 / Chapter 2.1.3 --- Discussion --- p.51 / Chapter 2.2 --- Expression of αMMC cDNA as an unfused protein --- p.52 / Chapter 2.2.1 --- Materials and methods --- p.52 / Chapter 2.2.1.1 --- Construction of the plasmid pET/MMC --- p.52 / Chapter 2.2.1.2 --- Preparation of αMMC insert by PCR --- p.56 / Chapter 2.2.1.3 --- Enzyme digestions --- p.57 / Chapter 2.2.1.4 --- Ligation --- p.58 / Chapter 2.2.1.5 --- Transformation --- p.59 / Chapter 2.2.1.6 --- Screening for αMMC inserts --- p.59 / Chapter 2.2.1.7 --- DNA sequencing --- p.60 / Chapter 2.2.1.8 --- Expression of unfused aMMC cDNA --- p.60 / Chapter 2.2.1.9 --- SDS-PAGE analysis --- p.61 / Chapter 2.2.1.10 --- Western blot analysis --- p.62 / Chapter 2.2.1.11 --- Purification of recombinant αMMC --- p.62 / Chapter 2.2.1.12 --- Biological activity of recombinant αMMC --- p.63 / Chapter 2.2.1.13 --- Radioimmunoassay --- p.63 / Chapter 2.2.2 --- Results --- p.67 / Chapter 2.2.2.1 --- Screening of pET/MMC --- p.67 / Chapter 2.2.2.2 --- DNA sequencing --- p.69 / Chapter 2.2.2.3 --- Expression of unfused αMMC cDNA --- p.69 / Chapter 2.2.2.4 --- Radioimmunoassay --- p.72 / Chapter 2.2.2.5 --- Purification of recombinant αMMC --- p.74 / Chapter 2.2.2.6 --- Biological activity of recombinant αMMC --- p.74 / Chapter 2.2.3 --- Discussion --- p.80 / Chapter CHAPTER 3 --- EXPRESSION OF MODIFIED FORMS OF αMMC cDNA --- p.82 / Chapter 3.1 --- Expression of deletion fragments of αMMC cDNA --- p.83 / Chapter 3.1.1 --- Materials and methods --- p.83 / Chapter 3.1.1.1. --- Construction of deletion mutants --- p.83 / Chapter 3.1.1.1.1 --- Modification of pRIT2T/MMC --- p.86 / Chapter 3.1.1.1.2 --- Preparation of closed circular DNA --- p.86 / Chapter 3.1.1.1.3 --- Alpha-phosphorothioate nucleotide --- p.87 / Chapter 3.1.1.1.4 --- Exo III digestion --- p.89 / Chapter 3.1.1.1.5 --- Ligation --- p.89 / Chapter 3.1.1.1.6 --- Transformation --- p.90 / Chapter 3.1.1.1.7 --- Screening of deletion subclones --- p.91 / Chapter 3.1.1.2 --- Confirmation of sequences --- p.91 / Chapter 3.1.1.3 --- Expression of deletion mutants --- p.92 / Chapter 3.1.1.4 --- Purification of deletion mutants --- p.92 / Chapter 3.1.1.5 --- Cleavage of deletion mutants --- p.93 / Chapter 3.1.1.6 --- Subcloning of the αMMC cDNA fragments --- p.94 / Chapter 3.1.1.7 --- Expression of the unfused deletion --- p.96 / Chapter 3.1.2 --- Results --- p.97 / Chapter 3.1.2.1 --- Designation of the deletion mutants --- p.97 / Chapter 3.1.2.2 --- Screening of deletion mutants --- p.98 / Chapter 3.1.2.3 --- DNA sequencing --- p.100 / Chapter 3.1.2.4 --- Expression of deletion mutants --- p.109 / Chapter 3.1.2.5 --- Purification of the fusion fragments --- p.111 / Chapter 3.1.2.6 --- Digestion of deletion mutants by factor Xa --- p.113 / Chapter 3.1.2.7 --- Subcloning of αMMC deletion fragments --- p.115 / Chapter 3.1.2.8 --- Expression of the unfused aMMC deletion --- p.117 / Chapter 3.1.3 --- Discussion --- p.119 / Chapter 3.2 --- Expression of a chimeric αMMC/TCS cDNA --- p.121 / Chapter 3.2.1 --- Materials and methods --- p.122 / Chapter 3.2.1.1 --- Construction of the MMC/TCS chimeric plasmid --- p.122 / Chapter 3.2.1.1.1 --- Digestion of pfG104 - Preparation of GH1100 --- p.125 / Chapter 3.2.1.1.2 --- Preparation of the GH405 fragment --- p.125 / Chapter 3.2.1.1.3 --- Digestion of pACYC177 --- p.126 / Chapter 3.2.1.1.4 --- "Dephosphorylation, ligation and transformation" --- p.126 / Chapter 3.2.1.1.5 --- Confirmation of insert orientation --- p.127 / Chapter 3.2.1.1.6 --- "Preparation of a fragment without PstI, ScaI" --- p.128 / Chapter 3.2.1.1.7 --- Preparation of the 750-bp TCS fragment --- p.128 / Chapter 3.2.1.1.8 --- Ligation of the TCS fragment --- p.129 / Chapter 3.2.1.1.9 --- Cleavage of pACYC177/TCS with ScaI and PstI --- p.129 / Chapter 3.2.1.1.10 --- Preparation of the PstI/HhaI-digested αMMC --- p.130 / Chapter 3.2.1.1.11 --- Ligation of the 252-bp fragment --- p.131 / Chapter 3.2.1.1.12 --- Cloning of MMC/TCS chimeric fragment --- p.131 / Chapter 3.2.1.2 --- Expression of pET/MMC-TCS --- p.132 / Chapter 3.2.1.3 --- SDS-PAGE analysis --- p.133 / Chapter 3.2.1.4 --- Western blot analysis --- p.134 / Chapter 3.2.1.5 --- Purification of MMC-TCS chimeric protein --- p.134 / Chapter 3.2.2 --- Results --- p.135 / Chapter 3.2.2.1 --- Construction of pET/MMC-TCS --- p.135 / Chapter 3.2.2.2 --- Expression of TCS/MMC chimeric cDNA --- p.140 / Chapter 3.2.2.3 --- Purification of MMC-TCS chimeric protein --- p.142 / Chapter 3.2.2.4 --- Reactivity of MMC-TCS chimeric protein with various antisera --- p.145 / Chapter 3.2.3 --- Discussion --- p.146 / Chapter CHAPTER 4 --- SCREENING OF αMMC IMMUNO-REACTIVE FRAGMENTS FROM A RANDOM FRAGMENT LIBRARY --- p.148 / Chapter 4.1 --- Materials and methods --- p.150 / Chapter 4.1.1 --- Description of the pTOPE vector --- p.150 / Chapter 4.1.2 --- Construction of an αMMC random fragment library --- p.152 / Chapter 4.1.2.1 --- Preparation of the cDNA insert of αMMC --- p.155 / Chapter 4.1.2.1.1 --- Large scale prearation of theE plasmid MMC18p8 --- p.155 / Chapter 4.1.2.1.2 --- Digestion of the plasmid MMC18p8 with EcoRI --- p.156 / Chapter 4.1.2.1.3 --- Electro-elution --- p.157 / Chapter 4.1.2.2 --- DNase I digestion --- p.158 / Chapter 4.1.2.3 --- Fractionation of DNA fragments --- p.159 / Chapter 4.1.2.3.1 --- Electrophoresis --- p.159 / Chapter 4.1.2.3.2 --- Electro-elution --- p.160 / Chapter 4.1.2.4 --- Single dA Tailing --- p.161 / Chapter 4.1.2.5 --- Ligation --- p.162 / Chapter 4.1.2.6 --- Transformation --- p.162 / Chapter 4.1.2.7 --- Controls --- p.163 / Chapter 4.1.2.7.1 --- Full-length αMMC cDNA control --- p.163 / Chapter 4.1.2.7.2 --- T-Vector ligation control --- p.164 / Chapter 4.1.2.8 --- Storage of the fragment library --- p.164 / Chapter 4.1.3 --- Immunoscreening of the random fragment library OF αMMC --- p.165 / Chapter 4.1.3.1 --- Anti-αMMC sera --- p.165 / Chapter 4.1.3.2 --- Purification of anti-αMMC sera --- p.165 / Chapter 4.1.3.3 --- Colony lift --- p.167 / Chapter 4.1.3.4 --- Induction of expression --- p.169 / Chapter 4.1.3.5 --- Colony lysis --- p.169 / Chapter 4.1.3.6 --- Immunoscreening --- p.170 / Chapter 4.1.4 --- PCR screening of inserts --- p.170 / Chapter 4.1.5 --- Amplification of positive signals --- p.172 / Chapter 4.1.6 --- Dot blot --- p.173 / Chapter 4.1.7 --- Confirmation of positive signals by Western blotting --- p.174 / Chapter 4.1.8 --- Analysis of positive clones by DNA sequencing --- p.175 / Chapter 4.1.9 --- Analysis of 3-dimensional structure of αMMC --- p.176 / Chapter 4.1.10 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of aMMC --- p.176 / Chapter 4.2 --- Results --- p.178 / Chapter 4.2.1 --- Theoretical considerations --- p.178 / Chapter 4.2.2 --- Construction of a random fragment library of αMMC cDNA --- p.180 / Chapter 4.2.3 --- Screening for immuno-reactive fragments of αMMC --- p.183 / Chapter 4.2.4 --- Confirmation of positive signals by Western blotting --- p.186 / Chapter 4.2.5 --- Estimation of fragment sizes by PCR --- p.188 / Chapter 4.2.6 --- Analysis of the fragment sequences --- p.190 / Chapter 4.2.7 --- Cross-reactivity of the immuno-reactive fragments --- p.194 / Chapter 4.2.8 --- Effect of a monoclonal anti-αMMC antibody (#A1) on ribosome-inactivating activity of αMMC --- p.196 / Chapter 4.3 --- Discussion --- p.198 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.200 / Concluding remarks --- p.214 / REFERENCES --- p.215 / APPENDIXES GENERAL PROCEDURES --- p.224 / Chapter A.l --- DNA sequencing --- p.224 / Chapter A.2 --- Purification of DNA with Gene Clean --- p.229 / Chapter A.3 --- Purification of primers after synthesis --- p.230 / Chapter A.4 --- Purification of plasmid DNA by Magic Prep (Promega) --- p.232 / Chapter A.5 --- Large-scale preparation of plasmid DNA by QIAGEN --- p.234 / Chapter A.6 --- Lowry protein determination --- p.236 / Chapter A.7 --- Preparation of acid phenol --- p.237 / Chapter A.8 --- SDS-polyacrylamide gel electrophoresis --- p.238
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318191 |
| Date | January 1994 |
| Contributors | Leung, Kwan-chi., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, iii, 240 leaves : ill. (some mounted col.) ; 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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