Poon, Chi-to, Geoffrey. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 115-125). / Acknowledgements --- p.I / Abbreviations --- p.II / Abstract --- p.III / Table of content --- p.IV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Importance of growth enhancement in aquaculture --- p.1 / Chapter 1.2 --- Physiological effect of growth hormone --- p.1 / Chapter 1.3 --- Progress in teleost growth hormone research --- p.3 / Chapter 1.4 --- Grass carp and its aquaculture --- p.5 / Chapter 1.5 --- Route of administration of growth hormone --- p.8 / Chapter 1.6 --- Nomenclature of baculovirus --- p.9 / Chapter 1.7 --- Biology of baculovirus --- p.10 / Chapter 1.8 --- Control of gene expression of virus-infected cells --- p.13 / Chapter 1.9 --- Theme of the thesis --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.18 / Chapter 2.1 --- Synthesis and purification of primers --- p.18 / Chapter 2.2 --- Modification of gcGH cDNA by polymerase chain reaction (PCR) --- p.20 / Chapter 2.3 --- TA cloning of PCR product --- p.20 / Chapter 2.4 --- Purification ofDNA fragment from agarose gel by GENECLEAN´ёØ --- p.21 / Chapter 2.5 --- Recovery of low molecular weight DNA fragment from agarose gel --- p.22 / Chapter 2.6 --- Small scale preparation of plasmid DNA --- p.23 / Chapter 2.7 --- Large scale plasmid preparation by QIAGEN´ёØ --- p.24 / Chapter 2.8 --- Preparation of competent Escherichia coli JM109 for transformation --- p.25 / Chapter 2.9 --- Transformation of plasmid into competent Escherichi coli JM109 --- p.26 / Chapter 2.10 --- Cell culture of BmN cell line --- p.26 / Chapter 2.10.1 --- Preparation of TC-100 insect medium --- p.27 / Chapter 2.10.2 --- Preparation of Grace's medium --- p.27 / Chapter 2.11 --- Extraction of wild-type Bombyx mori nuclear polyhedrosis virus DNA --- p.28 / Chapter 2.12 --- Transfection of BmN cells with Bombyx mori nuclear polyhedrosis virus DNA by DOTAP´ёØ --- p.28 / Chapter 2.13 --- Agarose plaque assay --- p.29 / Chapter 2.14 --- Lifting of vius plaque onto nitrocellulose filter paper --- p.30 / Chapter 2.15 --- Synthesis of radiolabelled DNA probe --- p.31 / Chapter 2.16 --- Pre-hybridization and hybridization of recombinant virus DNA on nitrocellulose paper --- p.31 / Chapter 2.17 --- Purification of recombinant virus by dot-blot manifold --- p.33 / Chapter 2.18 --- Preparation of cell lysate from virus-infected BmN cells --- p.33 / Chapter 2.19 --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.34 / Chapter 2.19.1 --- Staining of the gel by Coomassie blue method --- p.35 / Chapter 2.19.2 --- Staining of the gel by silver staining method --- p.35 / Chapter 2.20 --- Determination of protein concentration by Bradford's method --- p.36 / Chapter 2.21 --- Determination of total protein concentration by Folin-Lowry method --- p.36 / Chapter 2.22 --- Detection of grass carp growth hormone by Western blotting --- p.37 / Chapter 2.23 --- Preparation of native recombinant grass carp growth hormone for iodination --- p.38 / Chapter 2.24 --- Iodination of recombinat grass carp growth hormone by IODO-GEN´ёØ --- p.38 / Chapter 2.25 --- Purification of radiolabelled recombinant grass carp growth hormone --- p.39 / Chapter 2.26 --- Radioimmunoassay (RIA) for detection of recombinant grass carp growth hormone --- p.40 / Chapter 2.27 --- Ammonium sulphate precipitation --- p.41 / Chapter Chapter 3 --- Vector Construction --- p.42 / Chapter 3.1 --- Components of parent vector pBM030 --- p.42 / Chapter 3.2 --- Construction of pBM-EE --- p.44 / Chapter 3.3 --- Constrcution of pBM-EX --- p.47 / Chapter Chapter 4 --- Results --- p.51 / Chapter 4.1 --- Construction and purfication of recombinant baculovirus --- p.51 / Chapter 4.2 --- Expression of recombinant grass carp growth hormone in BmN cells --- p.55 / Chapter 4.3 --- Expression of recombinant grass carp growth hormone in Bombyx mori larva --- p.62 / Chapter 4.4 --- Putative physical characteristics of the recombinant grass carp growth hormone --- p.67 / Chapter 4.5 --- Purification of the grass carp growth hormone in Bombyx mori larva --- p.69 / Chapter 4.5.1 --- Ammonium sulphate precipitation --- p.69 / Chapter 4.5.2 --- Gel filtration --- p.72 / Chapter 4.5.3 --- Hydrophobic interaction chromatography --- p.75 / Chapter 4.5.4 --- Anion exchange chromatography --- p.78 / Chapter 4.5.5 --- Reverse phase chromatography --- p.90 / Chapter Chapter 5 --- Discussions --- p.99 / Chapter 5.1 --- Merits of baculovirus expression system against other expression systems --- p.99 / Chapter 5.2 --- Basic design of the recombinant baculovirus transfer vector --- p.100 / Chapter 5.3 --- Potential for Mutation of the Baculovirus during Homologous Recombination --- p.101 / Chapter 5.4 --- Cleavage of Signal Peptide from the Expressed Protein --- p.103 / Chapter 5.5 --- Difference in recombinant gcGH expression levelin EE4-7 and EX3-16 --- p.103 / Chapter 5.6 --- Purification of recombinant gcGH protein --- p.106 / Chapter 5.6.1 --- Chromatographic behaviour of recombinant gcGH in Q-Sepharose column --- p.106 / Chapter 5.6.2 --- Problem of aggregation of recombinant gcGH --- p.107 / Chapter 5.6.3 --- Solvent system used in recombinant gcGH purification --- p.108 / Chapter 5.6.4 --- Protein denaturating effect of the solvent system --- p.109 / Chapter 5.6.5 --- Protein yield --- p.110 / Chapter 5.7 --- Problems and accuracy of radioimmunoassay --- p.110 / Chapter Chapter 6 --- Further study --- p.113 / Chapter Chapter 7 --- References --- p.115 / Appendix I --- p.126 / Appendix II: Construction of the Supervector --- p.127
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318188 |
| Date | January 1994 |
| Contributors | Poon, Chi-to Geoffrey., 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, vii, 138 leaves : ill. (chiefly mounted) ; 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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