Poon Wing Ming Jodie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 207-233). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Abbreviations --- p.v / List of Figures --- p.x / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1. --- Severe Acute Respiratory Syndrome (SARS) --- p.1 / Chapter 1.1.1. --- Background of SARS --- p.1 / Chapter 1.1.2. --- Etiology and pathology of SARS --- p.3 / Chapter 1.1.3. --- Genome organization and expression of SARS-CoV --- p.5 / Chapter 1.1.4. --- Current molecular advances of SARS-CoV --- p.13 / Chapter 1.1.5. --- Current research advances on SARS-CoV nucleocapsid --- p.18 / Chapter 1.1.6. --- Current diagnostic assays of SARS-CoV infection --- p.23 / Chapter 1.1.7. --- Current treatment --- p.25 / Chapter 1.1.8. --- Vaccine development --- p.27 / Chapter 1.2. --- Aims of study --- p.30 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.33 / Chapter 2.1. --- Subcellular localization study of the SARS-CoV nucleocapsid protein --- p.33 / Chapter 2.1.1. --- "Cloning of SARS-CoV nucleocapsid cDNA into the green fluorescence protein (GFP) mammalian expression vector, pEGFP-C1" --- p.33 / Chapter 2.1.1.1. --- Amplification of SARS-CoV nucleocapsid gene by polymerase chain reaction (PCR) --- p.33 / Chapter 2.1.1.2. --- Purification of PCR products --- p.35 / Chapter 2.1.1.3. --- Restriction digestion of purified PCR products and the circular pEGFP-C 1 vector --- p.36 / Chapter 2.1.1.4. --- Ligation --- p.36 / Chapter 2.1.1.5. --- Preparation of chemically competent bacterial cell E.coli strain DH5a for transformation --- p.37 / Chapter 2.1.1.6. --- Transformation of ligation product into chemically competent bacterial cells --- p.38 / Chapter 2.1.1.7. --- Small-scale preparation of bacterial plasmid DNA --- p.39 / Chapter 2.1.1.8. --- Screening for recombinant clones --- p.40 / Chapter 2.1.1.9. --- DNA sequencing of cloned plasmid DNA --- p.41 / Chapter 2.1.1.10. --- Midi-scale preparation of recombinant plasmid DNA --- p.42 / Chapter 2.1.2. --- Cell culture --- p.44 / Chapter 2.1.2.1. --- Sub-culture of VeroE6 and HepG2 cell lines --- p.44 / Chapter 2.1.2.2. --- Transient transfection of GFP fusion construct --- p.45 / Chapter 2.1.3. --- Epi-fluorescent microscopy --- p.46 / Chapter 2.2. --- Study on differential gene expression patterns upon SARS-CoV nucleocpasid induction by cDNA microarray analysis --- p.48 / Chapter 2.2.1. --- Cloning of SARS-CoV N gene into mammalian expression vector pCMV-Tagl --- p.48 / Chapter 2.2.2. --- Cell culture --- p.50 / Chapter 2.2.2.1. --- Sub-culture of VeroE6 cell line --- p.50 / Chapter 2.2.2.2. --- Transient transfection of pCMV-Tag1 -SAR-CoV N construct --- p.50 / Chapter 2.2.3. --- Total RNA isolation --- p.51 / Chapter 2.2.3.1. --- Total RNA isolation by RNeasy Mini Kit --- p.51 / Chapter 2.2.3.2. --- Checking of RNA integrity --- p.53 / Chapter 2.2.3.3. --- Checking of RNA purity --- p.54 / Chapter 2.2.3.4. --- Determinations of total RNA concentrations and precipitation --- p.54 / Chapter 2.2.4. --- cDNA microarray (done by Affymetrix Inc. as a customer service) --- p.55 / Chapter 2.2.4.1. --- Precipitation of RNA --- p.55 / Chapter 2.2.4.2. --- Quantification of RNA --- p.56 / Chapter 2.2.4.3. --- Synthesis of double-stranded cDNA from total RNA --- p.56 / Chapter (i) --- First stand cDNA synthesis --- p.56 / Chapter (ii) --- Second cDNA synthesis --- p.57 / Chapter 2.2.4.4. --- Clean-up of double stranded cDNA --- p.58 / Chapter (i) --- Phase lock gel-phenol/ chloroform extraction --- p.58 / Chapter (ii) --- Ethanol precipitation --- p.58 / Chapter 2.2.4.5. --- Synthesis of biotin-labeled cRNA --- p.59 / Chapter 2.2.4.6. --- Clean-up and quantification of in vitro transcription (IVP) products --- p.59 / Chapter (i) --- In vitro transcription clean-up --- p.59 / Chapter (ii) --- Ethanol precipitation --- p.60 / Chapter (iii) --- Quantitation of cRNA --- p.60 / Chapter (iv) --- Sample checking --- p.60 / Chapter 2.2.4.7. --- cRNA fragmentation for target preparation --- p.60 / Chapter 2.2.4.8. --- Eukaryotic target hybridization --- p.61 / Chapter 2.2.4.9. --- "Probe array washing, staining and scanning" --- p.62 / Chapter 2.2.5. --- Confirmation of results by RT-PCR --- p.62 / Chapter 2.2.5.1. --- First-strand cDNA synthesis --- p.62 / Chapter 2.2.5.2. --- RT-PCR of candidate gene --- p.63 / Chapter 2.3. --- In vitro RNA interference of SARS-CoV nucleocapsid --- p.66 / Chapter 2.3.1. --- siRNA target site selection --- p.66 / Chapter 2.3.2. --- Cloning of target siRNA sequences into pSilencer 3.1-H1 vector --- p.71 / Chapter 2.3.3. --- Cell culture --- p.72 / Chapter 2.2.3.1. --- Sub-culture ofVeroE6 cells --- p.72 / Chapter 2.3.3.2. --- Transient co-transfection --- p.72 / Chapter 2.3.4. --- Detection of SARS-CoV nucleocapsid mRNA expression level by RT-PCR --- p.73 / Chapter 2.3.4.1. --- Total RNA isolation by TRIzol reagent --- p.73 / Chapter 2.3.4.2. --- First-strand cDNA synthesis --- p.74 / Chapter 2.3.4.3. --- RT-PCR assays --- p.74 / Chapter 2.3.5. --- Detection of SARS-CoV nucleocapsid protein expression level by Western blotting --- p.75 / Chapter 2.3.5.1. --- Total protein extraction --- p.75 / Chapter 2.3.5.2. --- Protein quantification --- p.75 / Chapter 2.3.5.3. --- Protein separation by SDS-PAGE and Western blot --- p.76 / Chapter 2.3.5.4. --- Western blot analysis --- p.78 / Chapter 2.4. --- Human fgl2 prothrombinase promoter analyses --- p.80 / Chapter 2.4.1. --- Cloning of the full-length human fgl2 prothrombinase promoter construct into a promoterless mammalian expression vector-pGL3-Basic --- p.80 / Chapter 2.4.2. --- Cloning of SARS-CoV Membrane gene into the mammalian expression vector pCMV-Tagl --- p.82 / Chapter 2.4.3. --- Cell culture --- p.84 / Chapter 2.4.3.1. --- Sub-culture of HepG2 and VeroE6 cell lines --- p.84 / Chapter 2.4.3.2. --- "Transient co-transfection of the full-length human fgl2 prothrombinase promoter construct with the pCMV-Tagl empty vector, pCMV-Tagl-SARS-CoV M expression vector, or pCMV-Tag1 -SARS-CoV N expression vector" --- p.84 / Chapter 2.4.4. --- Dual-luciferase reporter assay --- p.85 / Chapter 2.4.5. --- Detection of fgl2 mRNA expression level under the induction of SARS-CoV nucleocapsid protein by RT-PCR --- p.86 / Chapter 2.4.5.1. --- Total RNA isolation by TRIzol reagent --- p.86 / Chapter 2.4.5.2. --- First strand cDNA synthesis --- p.86 / Chapter 2.4.5.3. --- RT-PCR of fgl2 gene --- p.87 / Chapter CHAPTER THREE --- RESULTS --- p.88 / Chapter 3.1. --- Computer analysis of SARS-CoV Nucleocapsid --- p.88 / Chapter 3.2. --- Subcellular localization of SARS-CoV nucleopcasid protein in VeroE6 cells and HepG2 cells --- p.102 / Chapter 3.3. --- cDNA microarray analysis on differential gene expression pattern upon the over-expression of SARS-CoV Nucleocapsid gene --- p.114 / Chapter 3.4. --- In vitro RNA Interference of SARS nucleocapsid --- p.129 / Chapter 3.5. --- Transactivation of fgl2 prothrombinase gene promoter by SARS-CoV nucleocapsid protein in HepG2 and VE6 cells --- p.138 / Chapter CHAPTER FOUR --- DISCUSSION --- p.155 / Chapter 4.1. --- "The EGFP-tagged SARS-CoV N protein was localized in the cytoplasm only in VE6 cells, but translocated into both cytoplasm and nucleus in HepG2 cellsin the epi-fluorescence microscopy study" --- p.155 / Chapter 4.2. --- cDNA microarray demonstrated alternations of mRNA transcript level on a number of genes belonging to various functional classes upon over expression of SARS-CoV nucleocapsid gene --- p.162 / Chapter 4.3. --- RNA interference demonstrated effective gene silencing of SARS-CoV nucleocapsid gene --- p.171 / Chapter 4.4. --- SASR-CoV nucleocapsid protein induced the promoter activity of the prothrombinase fibrinogen-like protein2/ fibroleukin (fgl2) gene --- p.191 / Chapter 4.5. --- Conclusion --- p.196 / Chapter 4.6. --- Future work --- p.198 / Appendices --- p.199 / References --- p.207
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325535 |
| Date | January 2005 |
| Contributors | Poon, Wing Ming Jodie., 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, xix, 233 leaves : ill. ; 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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