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

In the past three decades, various steps of the human immunodeficiency virus type 1 (HIV-1) life cycle have been thoroughly studied. Many of these steps, such as viral entry, reverse transcription and proteolysis have been targets of antiretroviral therapy. Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity and this process appears to be chaperoned by the nucleocapsid (NC) protein of HIV-1. In this dissertation, the role of NC in genome dimerization and other aspects of the viral life cycle have been thoroughly studied. Various positions of the NC protein have been mutated through site-directed mutagenesis and relevant and dispensable positions of NC have been identified through this method. 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The various mutations introduced into NC show the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal or distal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch (plateau) or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein (Pr55gag). In the second section of my work, I studied the effect of Pr55gag processing on gRNA dimerization by introducing rate alternating mutants into Pr55gag protein cleavage sites. I showed that Maturation ofNCp15 into NCp9 is essential for fast rates of genomic RNA dimerization and maturation of NCp9 into NCp7 has no incidence on genomic RNA dimerization but is essential for viral replication. In order to delineate the amount of viral protease activity needed to produce mature virus 48 hours post transfection, we also studied, by cotransfection studies, the effect of various ratios of wild-type (BH10) and protease-inactive (PR- ) plasmids and found that HIV-1 reaches its full genomic RNA dimerization despite 75% unprocessed Pr55gag polyproteins. We have also shown that wild type BH10 plasmid can rescue those mutations in NCp7 protein that have an effect on gRNA dimerization through rescue experiments. Overall, this thesis sheds light on the role of NC in HIV-1 genome dimerization and other aspects of the viral life cycle and identifies the importance of each component of NC during these processes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

No description available.

by Chan Wai Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 170-185). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vii / Abbreviations --- p.xii / for Nucleotides --- p.xii / for Amino acids --- p.xii / for Standard genetic codes --- p.xiii / for Units --- p.xiii / for Prefixes of units --- p.xiv / for Terms commonly used in the report --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome --- p.1 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.3 --- Cell Biology of Coronavirus Infection and Replication and the Role of Nucleocapsid Protein --- p.9 / Chapter 1.4 --- Recent Advances in the SARS-CoV Nucleocapsid Protein --- p.16 / Chapter 1.5 --- The Sumoylation System --- p.24 / Chapter 1.6 --- Objectives of the Present Study --- p.28 / Chapter Chapter II --- SARS-CoV N protein and Fragment Purification --- p.29 / Chapter 2.1 --- INTRODUCTION --- p.29 / Chapter 2.2 --- METHODOLOGY --- p.31 / Materials --- p.31 / Methods --- p.39 / Chapter 2.2.1 --- Construction of the pMAL-c2P vector --- p.39 / Chapter 2.2.2 --- Sub-cloning of the N protein into expression vectors --- p.42 / Chapter 2.2.2.1 --- Design of primers for the cloning of N protein --- p.43 / Chapter 2.2.2.2 --- DNA amplification using Polymerase Chain Reaction (PCR) --- p.44 / Chapter 2.2.2.3 --- DNA extraction from agarose gel --- p.45 / Chapter 2.2.2.4 --- Restriction digestion of purified PCR product and vectors --- p.46 / Chapter 2.2.2.5 --- Ligation of N protein into expression vectors --- p.47 / Chapter 2.2.2.6 --- Preparation of competent cells --- p.48 / Chapter 2.2.2.7 --- Transformation of plasmids into competent Escherichia coli --- p.49 / Chapter 2.2.2.8 --- Preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.1 --- Mini-preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.2 --- Midi-preparation of plasmid DNA --- p.51 / Chapter 2.2.3 --- Expression of tagged and untagged N protein --- p.53 / Chapter 2.2.3.1 --- Preparation of E. coli competent cells for protein expression --- p.53 / Chapter 2.2.3.2 --- Expression of N protein --- p.53 / Chapter 2.2.3.3 --- Solubility tests on the fusion proteins expressed --- p.54 / Chapter 2.2.4 --- Purification of N protein Chromatographic methods --- p.55 / Chapter 2.2.4.1 --- Affinity chromatography --- p.55 / Chapter 2.2.4.1.1 --- Ni-NTA affinity chromatography --- p.55 / Chapter 2.2.4.1.2 --- Glutathione affinity chromatography --- p.56 / Chapter 2.2.4.1.3 --- Amylose affinity chromatography --- p.56 / Chapter 2.2.4.2 --- Ion exchange chromatography --- p.57 / Chapter 2.2.4.2.1 --- Cation exchange chromatography --- p.57 / Chapter 2.2.4.2.2 --- Anion exchange chromatography --- p.58 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.58 / Chapter 2.2.4.4 --- Size exclusion chromatography Purification strategies --- p.60 / Chapter 2.2.4.5 --- Purification of His6-tagged N proteins --- p.60 / Chapter 2.2.4.6 --- Purification of MBP-tagged N proteins --- p.60 / Chapter 2.2.4.7 --- Purification of GST-tagged N proteins --- p.61 / Chapter 2.2.4.8 --- Purification of untagged N proteins --- p.61 / Chapter 2.2.5 --- Trypsin digestion assay for the design of stable fragment --- p.64 / Chapter 2.2.6 --- Partial purification of the N protein amino acid residue 214-422 fragment --- p.65 / Chapter 2.2.7 --- Sumoylation of the SARS-CoV N protein --- p.67 / Chapter 2.2.7.1 --- In vitro sumoylation assay --- p.67 / Chapter 2.2.7.2 --- Sample preparation for mass spectrometric analysis --- p.68 / Chapter 2.3 --- RESULTS --- p.70 / Chapter 2.3.1 --- Construction of the vector pMAL-c2P --- p.70 / Chapter 2.3.2 --- "Construction of recombinant N protein-pAC28m, N-protein- pGEX-6P-l,N protein-pMAL-c2E and N protein-pMAL-c2P plasmids" --- p.72 / Chapter 2.3.3 --- Optimization of expression conditions --- p.79 / Chapter 2.3.4 --- Screening of purification strategies --- p.82 / Chapter 2.3.4.1 --- Purification of His6-N protein --- p.82 / Chapter 2.3.4.2 --- Purification of MBP-N protein --- p.84 / Chapter 2.3.4.3 --- Purification of GST-N protein --- p.85 / Chapter 2.3.4.4 --- Purification of untagged N protein --- p.87 / Chapter 2.3.5 --- Limited trypsinolysis for the determination of discrete structural unit --- p.91 / Chapter 2.3.6 --- Partial purification of the N protein 214-422 fragment --- p.94 / Chapter 2.3.7 --- Sumoylation of N protein --- p.97 / Chapter 2.2.7.1 --- Sumoylation site prediction --- p.97 / Chapter 2.2.7.2 --- In vitro sumoylation assay --- p.99 / Chapter 2.2.7.3 --- Mass spectrometric identification of sumoylated SARS-CoV N protein --- p.103 / Chapter 2.4 --- DISCUSSION --- p.109 / Chapter Chapter III --- Characterization of the Nucleic Acid Binding Ability of N protein --- p.119 / Chapter 3.1 --- INTRODUCTION --- p.119 / Chapter 3.2 --- METHODOLOGY --- p.120 / Materials --- p.120 / Methods --- p.124 / Chapter 3.2.1 --- Spectrophotometric Measurement of ratio OD260/ OD280 --- p.124 / Chapter 3.2.2 --- Native gel electrophoresis --- p.124 / Chapter 3.2.3 --- Quantitative determination of nucleic acids content --- p.125 / Chapter 3.2.3.1 --- Dische assay - quantitative determination of DNA content --- p.125 / Chapter 3.2.3.2 --- Orcinol assay - quantitative determination of RNA content --- p.126 / Chapter 3.2.4 --- RNase digestion of the N protein-bound RNA --- p.128 / Chapter 3.2.5 --- Isolation of RNA from purified GST-N proteins --- p.128 / Chapter 3.2.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.129 / Chapter 3.2.7 --- Vero E6 cell line maintenance and total RNA extraction --- p.131 / Chapter 3.2.8 --- Electrophoretic mobility shift assay (EMSA) --- p.131 / Chapter 3.3 --- RESULTS --- p.133 / Chapter 3.3.1 --- Detection of nucleic acids in the purified N proteins byspectrophotometric Measurement of ratio OD260/ OD280 --- p.133 / Chapter 3.3.2 --- Native gel electrophoresis --- p.135 / Chapter 3.3.3 --- Quantitative determination of nucleic acids content in purified GST-N proteins --- p.136 / Chapter 3.3.3.1 --- Dische assay for the determination of DNA --- p.136 / Chapter 3.3.3.2 --- Orcinol assay for the determination of RNA --- p.138 / Chapter 3.3.4 --- RNase digestion treatment --- p.139 / Chapter 3.3.5 --- Extraction of RNA from GST-N proteins --- p.140 / Chapter 3.3.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.142 / Chapter 3.3.7 --- Electrophoretic mobility shift assay (EMSA) --- p.144 / Chapter 3.4 --- DISCUSSION --- p.147 / Chapter Chapter IV --- Discussion --- p.154 / Chapter 4.1 --- "Purity, Aggregation and RNA Binding Property of the SARS-CoV Nucleocapsid Protein" --- p.154 / Chapter 4.2 --- Future perspectives --- p.156 / Chapter 4.2.1 --- Structural study of the SARS-CoV N protein through x-ray crystallography --- p.156 / Chapter 4.2.2 --- Mapping the RNA binding domain in the SARS-CoV N protein --- p.156 / Chapter 4.2.3 --- Determination of aggregation state by lateral turbidimetry analysis --- p.156 / Chapter 4.2.4 --- Exploring protein interacting partners that enhance RNA binding specificity --- p.157 / Appendix --- p.159 / Chapter I. --- Sequence of the SARS-CoV N protein --- p.159 / Chapter II. --- Sequence of the SARS-CoV genome fragment used for RNA binding assay in section 3.37.1 --- p.161 / Chapter III. --- Vector maps --- p.161 / Chapter a) --- Vector map of pACYC177 --- p.161 / Chapter b) --- Vector map and MCS of pET28a --- p.163 / Chapter c) --- Vector map and MCS of pAC28 --- p.164 / Chapter d) --- Vector map and MCS of pGEX-6P-1 / Chapter e) --- Vector map of pMAL-c2X and MCS of pMAL-c2E / Chapter IV. --- Electrophoresis markers --- p.166 / Chapter V. --- SDS-PAGE gel parathion protocol --- p.169 / References --- p.170

Wang Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 124-135). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Table of contents / List of figures / List of tables / List of abbreviations / CHAPTER / Chapter 1. --- Introduction / Chapter 1.1 --- Background of SARS and epidemiology / Chapter 1.2 --- SARS symptoms and infected regions / Chapter 1.3 --- SARS virus / Chapter 1.4 --- Treatment for SARS at present / Chapter 1.5 --- Vaccine development is a more effective way to fight against SARS / Chapter 1.6 --- Vaccine candidates / Chapter 1.6.1 --- Truncated S protein as a vaccine candidate / Chapter 1.6.2 --- Full-length N protein as a vaccine candidate / Chapter 1.7 --- E.coli expression system / Chapter 1.8 --- Baculovirus expression system / Chapter 1.8.1 --- Characteristics of baculovirus / Chapter 1.8.2 --- Infection cycle of baculovirus / Chapter 1.8.3 --- Control of viral gene expression in virus-infected cells / Chapter 1.8.4 --- Merits of baculovirus expression system / Chapter 1.9 --- Aim of study / Chapter 2. --- "Bacterial expression and purification of rS1-1000(E), rS401-1000(E) and rN(E)" / Chapter 2.1 --- Introduction / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Reagents for bacterial culture / Chapter 2.2.2 --- Reagents for agarose gel electrophoresis / Chapter 2.2.3 --- 2'-deoxyribonucleoside 5'-triphosphate (dNTP) mix for polymerase chain reaction (PCR) / Chapter 2.2.4 --- Sonication buffer / Chapter 2.2.5 --- Reagents for immobilized metal affinity chromatography (IMAC) purification / Chapter 2.2.6 --- Reagents for gel filtration chromatography / Chapter 2.2.7 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) / Chapter 2.2.8 --- Reagents for Western blotting / Chapter 2.3 --- Methods / Chapter 2.3.1 --- General techniques in molecular cloning / Chapter 2.3.2 --- "PCR amplification of the S1-400,S401-1000" / Chapter 2.3.3 --- Construction of clone pET-S 1-400 and PET-s401-1000 / Chapter 2.3.4 --- Construction of clone pAC-N / Chapter 2.3.5 --- Expression / Chapter 2.3.6 --- Inclusion bodies preparation / Chapter 2.3.7 --- Inclusion bodies solubilization using urea / Chapter 2.3.8 --- Protein refolding by rapid dilution and dialysis / Chapter 2.3.9 --- Purification of recombinant protein by nickel ion chelating Sepharose fast flow column (IMAC) / Chapter 2.3.10 --- Gel filtration chromatography for further purification / Chapter 2.3.11 --- Bradford assay for the protein concentration analysis / Chapter 2.3.12 --- Protein analysis / Chapter 2.4 --- Results / Chapter 2.4.1 --- SDS-PAGE analysis of the expressed proteins / Chapter 2.4.2 --- Western blot analysis of the bacterial cell lysate / Chapter 2.4.3 --- Protein purification by IMAC / Chapter 2.4.4 --- Purification of rS401-1000(E) by gel filtration / Chapter 2.4.5 --- Determination of production yield of recombinant fusion proteins / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- Expression vector selected for rS1-400(E) and rS401-1000(E) expression / Chapter 2.5.2 --- Protein expression in E.coli / Chapter 2.5.3 --- Purification process / Chapter 3. --- Baculovirus expression and purification of rS401-1000(ACN) and rN(BMN) protein / Chapter 3.1 --- Introduction / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Reagents for insect cell culture and virus work / Chapter 3.3 --- Methods / Chapter 3.3.1 --- "PCR amplification of N and cloning of S401-1000, N genes into the transfer vector pVL1393" / Chapter 3.3.2 --- Cloning of S401-1000 into transfer vector pFastBac HT B / Chapter 3.3.3 --- Virus works / Chapter 3.3.4 --- Identification of recombinant BmNPV or AcMNPV / Chapter 3.3.5 --- Manipulation of silkworm / Chapter 3.3.6 --- Mouse immunization for polyclonal antibody against rN(E) protein / Chapter 3.4 --- Results / Chapter 3.4.1 --- Expression of rN(BMN) in baculovirus / Chapter 3.4.2 --- Expression of rS401-1000(BMN) and rS401-1000(ACN) in baculovirus / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- The expression level of rN(BMN) in both in vitro and invivo / Chapter 3.5.2 --- The rS401-1000(ACN) protein expression level in vitro / Chapter 3.5.3 --- Failure in generating rS401-1000(BMN) / Chapter 3.5.4 --- Purification process of rN(BMN) by IMAC / Chapter 4. --- "Characterization of recombinant rS1-400(E), rN(E), rN(BMN), rS401_1000(E) and rS401-1000(ACN)" / Chapter 4.1 --- Introduction / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Reagents for enzyme-linked immunosorbent assay (ELISA) / Chapter 4.2.2 --- Reagents for purification of human IgG / Chapter 4.2.3 --- Source and identity of Immune sera / Chapter 4.3 --- Methods / Chapter 4.3.1 --- ELISA / Chapter 4.3.2 --- Purification process of human IgG / Chapter 4.4 --- Results / Chapter 4.4.1 --- Validation of Immune sera using SARS viral lysate / Chapter 4.4.2 --- Immunoreactivities of rS1-400(E) and rN(E) against pooled patients sera and normal human serum / Chapter 4.4.3 --- Immunoreactivity comparison of rN(E) and rN(BMN) / Chapter 4.4.4 --- Comparison of the immunoreactivities of rS401-1000(E) and rS401-1000(ACN) / Chapter 4.4.5 --- Immunoreactivity of SARS related proteins against Anti-SARS Antibody (Equine) / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Comparison of the immunoreactivities of SARS related proteins expressed in the present study / References

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV)&nbsp / showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a&nbsp / group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in&nbsp / children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of&nbsp / HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified&nbsp / ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie&nbsp / Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the&nbsp / production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the&nbsp / Western Cape.</p>

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV)&nbsp / showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a&nbsp / group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in&nbsp / children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of&nbsp / HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified&nbsp / ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie&nbsp / Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the&nbsp / production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the&nbsp / Western Cape.</p>

>Magister Scientiae - MSc / Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV) showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi ® vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the Western Cape. / South Africa