Chi-shing Lo. / Thesis submitted in: November 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 119-138). / Abstracts in English and Chinese. / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.v / Abstract --- p.vii / Abstract (Chinese version) --- p.ix / Table of contents --- p.xi / List of Figure --- p.xvii / List of Table --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- BIOLOGY OF TOXOPLASMA GONDII --- p.1 / Chapter 1.1.1 --- Life cycle of Toxoplasma gondii --- p.2 / Chapter (a) --- Tachyzoite --- p.3 / Chapter (b) --- Bradyzoite --- p.3 / Chapter 1.1.2 --- Genetics of Toxoplasma gondii --- p.4 / Chapter (a) --- Population genetics --- p.4 / Chapter (b) --- Molecular genetics --- p.5 / Chapter (c) --- Genome analysis --- p.7 / Chapter 1.1.3 --- Invasion --- p.8 / Chapter 1.1.4 --- Surface of Toxoplasma gondii --- p.9 / Chapter (a) --- Tachyzoite surface --- p.9 / Chapter (b) --- Bradyzoite surface --- p.11 / Chapter (c) --- Sporozite surface --- p.11 / Chapter (d) --- Glycoprotein antigens --- p.12 / Chapter 1.2 --- TREATMENT OF TOXOPLASMOSIS --- p.13 / Chapter 1.2.1 --- Chemotherapy --- p.13 / Chapter (a) --- Drug against metabolism and protein synthesis on nuclear genome --- p.13 / Chapter (b) --- Drug against other organelles --- p.14 / Chapter (c) --- Drug resistance --- p.15 / Chapter 1.2.2 --- Toxoplasma vaccine --- p.16 / Chapter (a) --- Mutant strains of Toxoplasma gondii as vaccine --- p.17 / Chapter (b) --- Subunit vaccine --- p.19 / Chapter (c) --- P30 as subunit vaccine --- p.20 / Chapter 1.3 --- AIM OF THE STUDY --- p.22 / Chapter Chapter 2 --- : Expression of P30 in Escherichia coli --- p.23 / Chapter 2.1 --- INTRODUCTION --- p.23 / Chapter 2.1.1 --- Why Escherichia coli? --- p.23 / Chapter 2.1.2 --- protein folding --- p.24 / Chapter 2.1.3 --- T7-based gene expression system --- p.25 / Chapter (a) --- Biology of T7 RNA polymerase --- p.26 / Chapter (b) --- pET translational vector --- p.26 / Chapter (c) --- Hislidine-tagged protein --- p.27 / Chapter (d) --- Host strain for expression --- p.28 / Chapter 2.2 --- MATERIALS --- p.29 / Chapter 2.2.1 --- Bactcrial strains --- p.29 / Chapter 2.2.2 --- Mouse strain --- p.29 / Chapter 2.2.3 --- Chemicals --- p.29 / Chapter 2.2.4 --- Nucleic acids --- p.30 / Chapter 2.2.5 --- Kit and reagents --- p.31 / Chapter 2.2.6 --- Antibodies --- p.31 / Chapter 2.2.7 --- Solutions --- p.32 / Chapter 2.2.8 --- Enzymes --- p.33 / Chapter 2.2.9 --- Sequencing primers --- p.33 / Chapter 2.3 --- METHODS --- p.34 / Chapter 2.3.1 --- Modification of P30 gene --- p.34 / Chapter (a) --- Preparation of recombinant plasmids，pBV220-ASP30PI and pBV220- SP30hisAPI --- p.36 / Chapter (b) --- Digestion of pBV220-ASP30PI and pBV220-SP30hisAPI with DraII and EcoRI --- p.37 / Chapter (c) --- Purification of DNA fragments from agarose gel --- p.37 / Chapter (d) --- Ligation of fragments of pBV220-ΔSP30PI and pBV220-SP30hisAPI --- p.38 / Chapter (e) --- Preparation of DH5α competent cells --- p.38 / Chapter (f) --- Transformation of recombinant pBV220-ΔSP30hisAPI --- p.38 / Chapter (g) --- Plasmid preparation of putative pBV220-ΔSP30API --- p.39 / Chapter (h) --- Plasmid preparation of pET-ΔSP30API --- p.39 / Chapter (i) --- Cycle sequencing reaction on putative plasmid pET-ASP30API --- p.40 / Chapter 2.3.2 --- Expression and Purification of his-tag P30 --- p.41 / Chapter (a) --- Expression profile of his-tag P30 production by IPTG induction --- p.41 / Chapter (b) --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.41 / Chapter (c) --- Purification of his-tag P30 --- p.43 / Chapter (d) --- Bradford Protein Microassay (Bio-Rad) --- p.43 / Chapter 2.3.3 --- Characterization of his-tag P30 --- p.44 / Chapter (a) --- Western blot of induced bacterial lysate by monoclonal anti-his-tag antibody --- p.44 / Chapter (b) --- Western blot of his-tag with seropositive sera of mice，rabbit and human --- p.46 / Chapter (c) --- Enterokinase digestion of his-tag P30 --- p.46 / Chapter (d) --- N'terminal amino acid sequencing of pure and enterokinase-cut his-tag --- p.47 / Chapter (e) --- Western blot of T. gondii lysate with antiserum against his-tag P30 --- p.47 / Chapter 2.4 --- RESULTS --- p.49 / Chapter 2.4.1 --- Modification of P30 gene --- p.49 / Chapter 2.4.2 --- "Expression, purification and characteriziation of his-tag P30 in bacteria" --- p.54 / Chapter 2.5 --- DISCUSSIONS --- p.64 / Chapter 2.5.1 --- Modification of P30 gene --- p.64 / Chapter 2.5.2 --- Expression and purification of his-tag P30 --- p.66 / Chapter 2.5.3 --- Characterization of his-tag P30 --- p.67 / Chapter Chapter 3 --- : Expression of P30 in Arabidopsis thalina --- p.69 / Chapter 3.1 --- INTRODUCTION --- p.69 / Chapter 3.1.1 --- Why Arabidopsis thalina? --- p.69 / Chapter 3.1.2 --- In planta transformation --- p.70 / Chapter 3.1.3 --- Transgenic plants as vacine production systems --- p.72 / Chapter (a) --- Stable expression of E. coli heat-liable enterotoxin B subunit and cholera-toxin B subunit --- p.73 / Chapter (b) --- Stable expression of Hepatitis B surface antigen (HBsAg) --- p.74 / Chapter (c) --- Stable expression of Norwalk virus capsid protein --- p.75 / Chapter (d) --- Transient expression by tobacco mosaic virus --- p.75 / Chapter (e) --- Transient expression by Cowpea mosaic virus capsid protein fusion --- p.76 / Chapter 3.2 --- MATERIALS --- p.77 / Chapter 3.2.1 --- Bacterial strains --- p.77 / Chapter 3.2.2 --- Arabidopsis strains --- p.77 / Chapter 3.2.3 --- Chemicals --- p.77 / Chapter 3.2.4 --- Nucleic acids --- p.78 / Chapter 3.2.5 --- Kit and reagents --- p.78 / Chapter 3.2.6 --- Solutions --- p.79 / Chapter 3.2.7 --- Enzymes and buffers --- p.81 / Chapter 3.2.8 --- PCR and Sequencing primers --- p.81 / Chapter 3.3 --- METHODS --- p.82 / Chapter 3.3.1 --- Construction of V7-ASP30API --- p.82 / Chapter 3.3.2 --- Agrobacterium-mediated transformation of Arabidopsis by vacuum infiltration --- p.83 / Chapter (a) --- Preparation of electro-competent Agrobacterium --- p.83 / Chapter (b) --- Transformation of electro-competent Agrobacterium with V7- ASP30API --- p.84 / Chapter (c) --- Plasmid preparation of V7-ASP30API from transformed Agrobacterium --- p.84 / Chapter (d) --- Vacuum infiltration --- p.85 / Chapter 3.3.3 --- Screening of homozygous transgenic plants --- p.86 / Chapter 3.3.4 --- Detecton of transgene P30 in genomic DNA of transgenic plants --- p.87 / Chapter (a) --- Preparation of DIG-labelled probe --- p.87 / Chapter (b) --- Estimation the yield of DIG-labelled probe --- p.88 / Chapter (c) --- Extraction of genomic DNA from transgenic plants --- p.88 / Chapter (d) --- Restriction digestion of genomic DNA with EcoRI and HindIII --- p.89 / Chapter (e) --- DNA transfer from gel to nylon membrane --- p.89 / Chapter (f) --- Detection of hybridized DIG-labelled probe on membrane/ blot --- p.90 / Chapter (g) --- PCR on genomic DNA of transgenic plants with specific primers --- p.91 / Chapter 3.3.5 --- Analysis of transgene RNA expression in transgenic plants --- p.91 / Chapter (a) --- Extraction of total RNA from plants --- p.91 / Chapter (b) --- Northern blot on RNA of F2 transgenic plants --- p.92 / Chapter (c) --- RT-PCR on RNA of F3 transgenic plants --- p.93 / Chapter 3.3.6 --- Detection of his-tag P30 protein in F3 transgenic plants --- p.93 / Chapter 3.4 --- RESULTS --- p.95 / Chapter 3.4.1 --- Construction of V7-ASP30API --- p.95 / Chapter 3.4.2 --- Screening of homozygous transgenic plants --- p.99 / Chapter 3.4.3 --- Molecular analysis of transgene P30 in transgenic plants --- p.101 / Chapter 3.5 --- DISCUSSIONS --- p.108 / Chapter 3.5.1 --- Construction and optimization of expression construct --- p.108 / Chapter 3.5.2 --- Screening and selection of homozyous transgenic plants --- p.109 / Chapter 3.5.3 --- Analysis of transgenic plants --- p.110 / Chapter Chapter 4 : --- General Discussions --- p.112 / Chapter 4.1 --- Significances of studying Toxoplasma gondii --- p.112 / Chapter 4.2 --- Expression of recombinant P30 in prokaryotic systems --- p.113 / Chapter 4.2 --- Expression of recombinant P30 in eukaryotic systems --- p.115 / Reference --- p.119