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Expression and purification of recombinant grass carp (ctenopharyngodon idellus) growth hormone in BmN cells and silkworm (bombyx mori) larvae.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318188
Date January 1994
ContributorsPoon, Chi-to Geoffrey., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
PublisherChinese University of Hong Kong
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, vii, 138 leaves : ill. (chiefly mounted) ; 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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