No description available.

No description available.

There are an estimated 350 million people chronically infected with Hepatitis B Virus (HBV), of which approximately 600 000 die each year from HBV complications including cirrhosis and liver cancer. The X protein from HBV (HBx) has been implicated in the progression of chronic HBV to liver cancer and has been reported to manipulate several critical cellular pathways. These include the cell cycle, the tumour suppressor protein p53, protein degradation and signal transduction pathways. The role of these interactions in HBV replication and the viral lifecycle is currently unknown. The lack of animal models and infectable cell lines together with solubility and stability issues related to the HBx protein have made progress difficult. The reliance on approximate cellular and animal models has yielded many discordant studies that have confounded our interpretations of the role of HBx. There have been no novel approaches attempting to express HBx at a quantity and quality sufficient for high resolution X-ray and nuclear magnetic resonance structural determination. Additionally no bioinformatic analyses have been applied to HBx, and thus distinctive features of HBx that may be responsible for these challenges have not been reported. This thesis describes the detailed experimentation to express and purify HBx in a functional, soluble and stable form. The study focussed on Saccharomyces cerevisiae and Semliki Forest Virus (SFV) expression systems, together with the use of a solubility-enhancing Maltose Binding Protein protein tag (MBP). The S. cerevisiae-based pYES2 and YEp and mammalian expression vectors showed production of HBx protein. However HBx that had been expressed using S. cerevisiae and human cells could not be reliably detected in Western blots using antibodies raised against E. coliexpressed HBx. This result was despite the positive visualisation of HBx using the same antibodies and immunofluorescence microscopy. This validated previous reports describing the variable antigenicity of HBx. Furthermore these findings supported the decision to develop eukaryotic-based HBx expression vectors as results suggested structural differences between eukaryote and prokaryote expressed protein. HBx was subsequently detected and purified in a soluble and active form using an MBP tag as well as a SFV expression vector. All of these options provide an excellent point from which further work at optimising HBx expression and structural elucidation can occur. Bioinformatic analysis of HBx suggested the presence of protein disorder and protease sensitive sites within the negative regulatory domain of HBx. Literature descriptions of the molecular promiscuity that protein disorder allows, offers an explanation for the presence of the discordant findings on HBx interactions and functions. It is generally accepted that proteins containing disorder are tightly regulated and thus experimental systems employing overexpression methodologies may encourage cellular toxicity and non-specific interactions through the use of short linear motifs. Evolutionary analysis of HBx sequences revealed that the eight HBV genotypes (A-H) showed concordance regarding synonymous and non-synonymous substitutions at the overlapping and non-overlapping domains of hbx. Substitutions in hbx were most common at positions where a synonymous substitution occurred in the overlapping partner gene. The presence of sites under positive, neutral and negative selection were identified across the length of HBx. The different genotypes showed positive selection indicating selective pressures unique to each, thus offering a contributing explanation for the variable disease severity observed between the subtypes. Overall, this thesis has provided novel methods to express and purify HBx in S. cerevisiae and mammalian cells. These methods, together with an increased understanding of the nature of HBx sequences through bioinformatic analysis, pave the way to conduct both structural studies and biological assays to elucidate the genuine roles of HBx in the HBV lifecycle and its contribution to the progression to liver cancer.

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

Not available / text

No description available.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisors: Angela M. Belcher and David A. Vanden Bout. Vita. Includes bibliographical references. Also available from UMI.

No description available.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: CTL; dengue. Includes bibliographical references (p. 57-64).

Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.