by Cheung Yeuk Siu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-153). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 摘要 --- p.iv / List of abbreviations --- p.v / Table of contents --- p.viii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prolactin (PRL) --- p.1 / Chapter 1.1.1 --- General introduction --- p.1 / Chapter 1.1.2 --- Genomic organization of teleost PRL gene --- p.2 / Chapter 1.1.3 --- Conserved domains of fish PRL --- p.3 / Chapter 1.1.4 --- Structure of teleost PRL --- p.6 / Chapter 1.1.5 --- Tissue sources of PRL --- p.8 / Chapter 1.2 --- Prolactin receptor (PRLR) --- p.9 / Chapter 1.2.1 --- Tissue distribution in teleosts --- p.9 / Chapter 1.2.2 --- Receptor structure and multiple forms of PRLR --- p.11 / Chapter 1.2.3 --- Possible action mechanisms of PRL --- p.14 / Chapter 1.3 --- Control of PRL release --- p.17 / Chapter 1.4 --- Biological functions of PRL in vertebrates --- p.19 / Chapter 1.4.1 --- Biological effects on teleosts --- p.19 / Chapter 1.4.1.1 --- Osmoregulatory roles --- p.20 / Chapter 1.4.1.2 --- Non-osmoregulatory roles --- p.29 / Chapter 1.5 --- Aim of the present study --- p.32 / Chapter Chapter 2 --- "Recombinant goldfish (Carassius auratus) prolactin: subcloning, expression, purification and refolding of the recombinant protein" / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.3 --- Methods --- p.48 / Chapter 2.3.1 --- Subcloning of the gfPRL cDNA --- p.48 / Chapter 2.3.1.1 --- PCR cloning of gfPRL cDNA --- p.48 / Chapter 2.3.1.2 --- DNA sequencing of the subcloned fragment --- p.51 / Chapter 2.3.1.3 --- Subcloning of the gfPRL cDNA fragment into the expression vector --- p.52 / Chapter 2.3.2 --- Expression and purification of rgfPRL --- p.53 / Chapter 2.3.2.1 --- Transformation of pRSETA/gfPRL into BL21(DE3)pLysS cells --- p.53 / Chapter 2.3.2.2 --- Prokaryotic expression of rgfPRL --- p.53 / Chapter 2.3.2.3 --- Affinity purification of rgfPRL --- p.55 / Chapter 2.3.2.4 --- Western blot analysis of the purified rgfPRL --- p.57 / Chapter 2.3.2.5 --- Protein concentration determination of the rgfPRL --- p.59 / Chapter 2.3.3 --- Protein refolding --- p.60 / Chapter 2.4 --- Results --- p.61 / Chapter 2.4.1 --- Subcloning and DNA sequencing of the gfPRL --- p.61 / Chapter 2.4.2 --- Expression and purification of rgfPRL --- p.63 / Chapter 2.4.2.1 --- Prokaryotic expression of rgfPRL --- p.63 / Chapter 2.4.2.2 --- Affinity purification of rgfPRL --- p.66 / Chapter 2.4.2.3 --- Western blot analysis of the purified rgfPRL --- p.68 / Chapter 2.4.2.4 --- Protein concentration determination of the rgfPRL --- p.68 / Chapter 2.4.3 --- Protein refolding --- p.70 / Chapter 2.5 --- Discussion --- p.72 / Chapter Chapter 3 --- Production of polyclonal antibodies against rgfRPL / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials --- p.82 / Chapter 3.3 --- Methods --- p.84 / Chapter 3.3.1 --- Immunization of rabbits --- p.84 / Chapter 3.3.2 --- Collection of the polyclonal antisera --- p.85 / Chapter 3.3.3 --- Purification of IgG from the polyclonal antisera --- p.86 / Chapter 3.3.4 --- Enzyme linked immunosorbent assay (ELISA) --- p.87 / Chapter 3.3.5 --- Western blot analysis for cross-reactivity --- p.88 / Chapter 3.4 --- Results --- p.90 / Chapter 3.4.1 --- Isolation and purification of IgG from the polyclonal antisera --- p.90 / Chapter 3.4.2 --- ELISA --- p.93 / Chapter 3.4.3 --- Western blot analysis for cross-reactivity --- p.96 / Chapter 3.5 --- Discussion --- p.98 / Chapter Chapter 4 --- Isolation of native PRL from goldfish pituitaries / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Materials --- p.101 / Chapter 4.3 --- Methods --- p.103 / Chapter 4.3.1 --- Alkaline extraction --- p.103 / Chapter 4.3.2 --- Size exclusion chromatography --- p.104 / Chapter 4.3.3 --- Anion exchange chromatography --- p.104 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Size exclusion chromatography --- p.106 / Chapter 4.4.2 --- Anion exchange chromatography --- p.109 / Chapter 4.4.3 --- SDS-PAGE analysis and immuno-detection of the purified protein --- p.112 / Chapter 4.5 --- Discussion --- p.114 / Chapter Chapter 5 --- Receptor binding assays / Chapter 5.1 --- Introduction --- p.115 / Chapter 5.2 --- Materials --- p.117 / Chapter 5.3 --- Methods --- p.119 / Chapter 5.3.1 --- Gill membrane preparation --- p.119 / Chapter 5.3.2 --- Radioactive labelling of the primary ligand --- p.120 / Chapter 5.3.3 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.121 / Chapter 5.3.4 --- Membrane protein dependence assay --- p.122 / Chapter 5.3.5 --- Receptor binding study using rgfPRL --- p.124 / Chapter 5.4 --- Results --- p.125 / Chapter 5.4.1 --- Radioactive labelling of the primary ligand --- p.125 / Chapter 5.4.2 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.127 / Chapter 5.4.3 --- Membrane protein dependence assay --- p.129 / Chapter 5.4.4 --- Receptor binding study using rgfPRL --- p.131 / Chapter 5.5 --- Discussion --- p.133 / Chapter Chapter 6 --- General discussion and conclusion --- p.136 / References --- p.141
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323158 |
| Date | January 2000 |
| Contributors | Cheung, Yeuk Siu., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xii, 153 leaves : ill. (some 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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