by Chung Ka Kin Kenny. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 116-120). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iii / Abbreviations --- p.viii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Chemical and Biological Properties of TCS --- p.1 / Chapter 1.1.1 --- Chemical properties --- p.2 / Chapter 1.1.2 --- Biological properties --- p.5 / Chapter 1.2 --- Structure of Antigens --- p.6 / Chapter 1.3 --- Methods Used for the Mapping of Antigenic Sites on the Protein --- p.8 / Chapter 1.4 --- Objective and Strategy of the Study of the Antigenic Determinants of TCS --- p.13 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Bacterial Strains Used --- p.15 / Chapter 2.2 --- General Techniques --- p.16 / Chapter 2.2.1 --- Extraction of DNA by phenol/chloroform --- p.16 / Chapter 2.2.2 --- Ethanol precipitation --- p.16 / Chapter 2.2.3 --- Minipreparation of plasmid --- p.16 / Chapter 2.2.4 --- Preparation of plasmid DNA using Qiagen pack 100 Cartridge --- p.17 / Chapter 2.2.5 --- Preparation of plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.18 / Chapter 2.2.6 --- Preparation and transformation of Escherichia coli competent cells --- p.19 / Chapter 2.2.7 --- Agarose gel electrophoresis of DNA --- p.21 / Chapter 2.2.8 --- Restriction enzyme digestion of DNA --- p.21 / Chapter 2.2.9 --- Purification of DNA fragment from agarose gel using Gene Clean® kit --- p.22 / Chapter 2.2.10 --- Ligation of insert into vector --- p.22 / Chapter 2.2.11 --- Rapid screening for the presence of plasmid by direct lysis of the bacterial colony --- p.23 / Chapter 2.2.12 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.24 / Chapter 2.2.13 --- Staining of protein in polyacrylamide gel --- p.25 / Chapter 2.2.14 --- Western blot detection of TCS --- p.25 / Chapter 2.2.15 --- Autoradiography --- p.27 / Chapter 2.3 --- Production of Recombinant Anti-TCS Fab Fragment --- p.27 / Chapter 2.3.1 --- Cloning and screening of the recombinant anti-TCS Fab fragment --- p.27 / Chapter 2.3.2 --- Expression of recombinant anti-TCS Fab fragment --- p.28 / Chapter 2.4 --- Construction of Deletion Library of pET58210 by Transposon Mediated Mutagenesis --- p.29 / Chapter 2.5 --- PCR Insertion Mapping of the Transposon Inserted Clones --- p.31 / Chapter 2.6 --- DNA Sequencing of Positive Clones After the PCR Insertion Mapping --- p.32 / Chapter 2.6.1 --- DNA sequencing reaction --- p.33 / Chapter 2.6.2 --- DNA sequencing gel casting and electrophoresis --- p.34 / Chapter 2.7 --- Computer Analysis of the DNA Sequence --- p.35 / Chapter 2.8 --- Subcloning of the Positive Clone that have been Located by DNA Sequencing --- p.35 / Chapter 2.8.1 --- Subcloning of the mutated TCS gene sequence with the Tn1000 delta end facing the N-tenninal of the TCS gene sequence --- p.36 / Chapter 2.8.2 --- Subcloning of the mutated TCS gene sequence with the Tn1000 gamma end facing the N-terminal of the TCS gene sequence --- p.37 / Chapter 2.9 --- Construction of Deletion Mutants that Exclude the 21 Amino Acids in the N-terminal of TCS --- p.39 / Chapter 2.10 --- Overexpression of Mutated TCS Protein with the T7 RNA Polymerase Driven Expression System --- p.41 / Chapter 2.11 --- Analysis of TCS Deletion mutants by Western Blotting --- p.42 / Chapter 2.12 --- Preparation of Recombinant TCS Protein --- p.42 / Chapter 2.13 --- Proteolytic and Chemical Cleavage of Recombinant TCS --- p.44 / Chapter 2.14 --- Tricine-SDS-16.5% Polyacrylamide Gel Electrophoresis --- p.45 / Chapter 2.15 --- Electroblotting Using Polyvinylidene Difluoride Membrane (PVDF) for N-terminal Determination --- p.48 / Chapter 2.16 --- Reagents and Buffers --- p.49 / Chapter 2.16.1 --- Media for bacterial culture --- p.49 / Chapter 2.16.2 --- Reagents for preparation of plasmid DNA --- p.51 / Chapter 2.16.3 --- DNA electrophoresis buffers --- p.51 / Chapter 2.16.4 --- Reagents for SDS-PAGE --- p.52 / Chapter 2.16.5 --- Reagents for Western blot --- p.53 / Chapter 2.16.6 --- Reagents for DNA sequencing --- p.54 / Chapter 2.16.7 --- Reagents for electroblotting using PVDF --- p.54 / Chapter Chapter 3 --- The Study of Antigenic Determinants of TCS by Transposon Mediated Deletion Mutagenesis / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Construction of deletion library of pET58210 by transposon mediated mutagenesis --- p.56 / Chapter 3.2.2 --- PCR insertion mapping of the transposon inserted clones --- p.60 / Chapter 3.2.3 --- DNA sequencing of positive clones after the PCR insertion mapping --- p.63 / Chapter 3.2.4 --- Computer analysis of the DNA sequence --- p.65 / Chapter 3.2.5 --- Expression of mutated plasmids with Tn 1000 --- p.65 / Chapter 3.2.6 --- Subcloning of the positive clones that have been characterized by DNA sequencing --- p.68 / Chapter 3.2.7 --- Overexpression of mutated TCS protein with the T7 RNA polymerase driven expression system --- p.71 / Chapter 3.2.8 --- Analysis of TCS deletion mutants by Western blotting --- p.71 / Chapter 3.2.9 --- Construction of deletion mutants that exclude the 21 amino acids in the N-terminal of TCS --- p.77 / Chapter 3.3 --- Discussion --- p.80 / Chapter Chapter 4 --- The Study of Antigenic Determinants on TCS by Chemical and Enzymatic Cleavages of TCS Protein / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Results --- p.91 / Chapter 4.2.1 --- Preparation of recombinant TCS protein --- p.91 / Chapter 4.2.2 --- Proteolytic and chemical cleavages of recombinant TCS --- p.94 / Chapter 4.2.3 --- Western blotting analysis of TCS protein fragment generated by proteolytic and chemical cleavage --- p.96 / Chapter 4.2.4 --- N-terminal determination of the antigenic TCS fragment produced by chemical cleavage --- p.100 / Chapter 4.3 --- Discussion --- p.103 / Chapter Chapter 5 --- General Discussion and Future Study / Chapter 5.1 --- General Discussion --- p.107 / Chapter 5.2 --- Future Study --- p.115 / References --- p.116
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318228 |
| Date | January 1994 |
| Contributors | Chung, Ka Kin Kenny., 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, viii, 120 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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