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Expression and characterization of SARS spike and nucleocapsid proteins and their fragments in baculovirus and E.coli. / Expression & characterization of SARS spike and nucleocapsid proteins and their fragments in baculovirus and E.coli

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325167
Date January 2005
ContributorsWang, Ying., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xx, 149 leaves : ill. (some col.) ; 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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