Expression of a synthetic gene encoding a multi-epitopic antigen for toxoplasma gondii.

Ng Wai-yan.Thesis submitted in: September 2003.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 200-225).Abstracts in English and Chinese.Statement --- p.iiiAcknowledgments --- p.ivAbbreviations --- p.vAbstract --- p.viiAbstract (Chinese version) --- p.ixTable of contents --- p.xiList of Figure --- p.xxList of Table --- p.xxiiiChapter Chapter 1 --- : General Introduction --- p.1Chapter 1.1 --- Toxoplasma gondii and Toxoplasmosis --- p.1Chapter 1.1.1 --- Biology and life cycle of Toxoplasma gondii --- p.2Chapter 1.2 --- Treatment of Toxoplasmosis --- p.4Chapter 1.2.1 --- Chemotherapy --- p.4Chapter (a) --- Pyrimethamine and sulfadiazine --- p.4Chapter (b) --- Clindamycin and Spiramycin --- p.4Chapter (c) --- Hydroxynaphthoquinones and new Macrolides --- p.5Chapter 1.2.2 --- Toxoplamsa Vaccine --- p.5Chapter (a) --- Vaccine using mutant strain of T. gondii --- p.6Chapter (b) --- Subunit vaccine --- p.6Chapter 1.3 --- The Immune Responses --- p.8Chapter 1.3.1 --- Protective immune responses --- p.8Chapter (a) --- Cytokines involved in the immunity against T. gondii --- p.10IFN-γ: Role in resistance to infection --- p.10TNF-α: Synergetic Role with IFN-γ --- p.10IL-12: Role in T cell differentiation --- p.11IL-10: Role in immuno-regulation --- p.12Other cytokines --- p.12Chapter (b) --- Immunomodulatory role of Nitric oxide (NO) --- p.13Chapter (c) --- Humoral immunity against T. gondii --- p.13Chapter 1.3.2 --- Toxoplasma proteins known to elicit T cell-dependent immunity --- p.14Chapter (a) --- Surface antigen 1 (SAG1/ P30) --- p.16Chapter (b) --- Dense granules protein 2 (GRA2/ P28) --- p.17Chapter (c) --- Rhoptry protein 2 (ROP2/ P54) --- p.18Chapter 1.3.3 --- Appropriate vaccine design --- p.18Chapter 1.4 --- Aim of the study --- p.23Chapter Chapter 2 ： --- Expression of the Synthetic Gene Encoding the Multi-epitopic Antigen (MEA) for Toxoplasma gondii in Escherichia coli --- p.25Chapter 2.1 --- Introduction --- p.25Chapter 2.1.1 --- Multi-epitopic antigen for T. gondii as vaccine --- p.26Chapter a) --- "Epitopes from Toxoplasma antigens, P30, GRA2 and ROP2" --- p.26"T-cell epitopes from P30 (aa 138-154, aa 191-209)" --- p.26T-cell epitope from ROP2 (aa 197-216) --- p.27T-and B-cell epitope from GRA2 (aa 171-185) --- p.27Chapter b) --- Helper T-cell epitope from tetanus toxin --- p.27Chapter c) --- Linker --- p.28Chapter 2.1.2 --- Escherichia coli Expression System --- p.31Chapter (a) --- The T7 RNA polymerase and T7 lac Promoter --- p.31Chapter (b) --- Other translational elements --- p.32Chapter (c) --- Fusion partner --- p.33Chapter (d) --- Choice of expression host strain --- p.33Chapter 2.2 --- Materials --- p.35Chapter 2.2.1 --- Bacterial strains --- p.35Chapter 2.2.2 --- Mouse strains --- p.35Chapter 2.2.3 --- Chemicals --- p.35Chapter 2.2.4 --- Nucleic acids --- p.36Chapter 2.2.5 --- Kits and reagents --- p.37Chapter 2.2.6 --- Antibodies --- p.37Chapter 2.2.7 --- Solutions --- p.38Chapter 2.2.8 --- Enzymes --- p.40Chapter 2.2.9 --- Primers --- p.40Chapter 2.3 --- Methods --- p.41Chapter 2.3.1 --- Design and synthesis of the synthetic gene encoding the multi-epitopic antigen for Toxoplasma gondii --- p.41Chapter 2.3.2 --- Cloning of the MEA into the E. coli expression vector --- p.41Chapter (a) --- Preparation of plasmid : pCRII-TOPO-MEA and pET30a+ --- p.43Chapter (b) --- PCR amplification of MEA from pCRII-TOPO-MEA --- p.44Chapter (c) --- Digestion and purification of the E. coli expression vector pET30a+ with Ncol --- p.44Chapter (d) --- Fill-in Ncol cut pET30a+ --- p.44Chapter (e) --- Purification of DNA fragment from agarose gel --- p.45Chapter (f) --- "Ligation of MEA fragment and Ncol cut, fill-in pET30a+" --- p.45Chapter (g) --- Preparation of DH5a competent cells --- p.46Chapter (h) --- Transformation of recombinant pET30a+-MEA --- p.46Chapter (i) --- PCR screening and plasmid preparation for the putative pET30a+- HisMEA --- p.46Chapter (j) --- Cycle sequencing reaction on putative plasmid pET30a+-MEA --- p.47Chapter 2.3.3 --- Expression and purification of his-tag MEA --- p.48Chapter (a) --- Expression profile of His-tag MEA production by IPTG induction --- p.48Chapter (b) --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.49Chapter (c) --- Estimation of His-tag MEA production in induced bacterial lysate --- p.50Chapter (d) --- Purification of His-tag MEA --- p.50Chapter (e) --- Braford Protein Microassay (Bio-rad) --- p.50Chapter 2.3.4 --- Characterization of his-tag MEA --- p.51Chapter (a) --- Western blot of induced bacteriallysate by monoclonal anti-his-tag antibody --- p.52Chapter (b) --- N'-terminal amino acid sequencing of His-tag MEA --- p.52Chapter (c) --- Western blot of His-tag MEA with seropositive serum of mice and human --- p.53Chapter (d) --- Preparation of Toxoplasma gondii lysate --- p.54Chapter (e) --- Western blot of purified his-tag MEA and T. gondii Lysate by anti- serum of recombinant his-tag MEA --- p.54Chapter 2.4 --- Results --- p.56Chapter 2.4.1 --- Design and synthesis of the synthetic gene encoding the multi- epitopic antigen for Toxoplasma gondii --- p.56Chapter 2.4.2 --- Cloning of MEA into E. coli expression vector --- p.59Chapter 2.4.3 --- Expression and purification of His-tag MEA --- p.61Chapter 2.4.4 --- Charterization of His-tag MEA --- p.67Chapter 2.5 --- Discussion --- p.73Chapter 2.5.1 --- Design and synthesis of the synthetic gene encoding the multi-epitopic antigen for Toxoplasma gondii --- p.73Chapter 2.5.2 --- Cloning of MEA into E. coli expression vector --- p.77Chapter 2.5.3 --- Expression and purification of His-tag MEA --- p.77Chapter 2.5.3 --- Characterization of His-tag MEA --- p.78Chapter Chapter 3: --- Immunological Studies of the Multi-epitopic Antigen (MEA) for Toxoplasma gondii in Mouse Models --- p.82Chapter 3.1 --- Introduction --- p.82Chapter 3.1.1 --- "Expression of P30, GRA2 and ROP2 in E. coli expression system" --- p.82Chapter (a) --- Expression of P30 --- p.82Chapter (b) --- Expression of ROP2 --- p.83Chapter (c) --- Expression of GRA2 --- p.84Chapter 3.1.2 --- Immunizations --- p.84Chapter (a) --- Choices of animals --- p.84Chapter (b) --- Adjuvants --- p.85Chapter 3.1.3 --- Measurements of cellular immune responses in vaccinated mice --- p.86Chapter 3.2 --- Materials --- p.87Chapter 3.2.1 --- Mouse strains --- p.87Chapter 3.2.2 --- Chemicals --- p.87Chapter 3.2.3 --- "Culture Medium, buffer and other solutions" --- p.87Chapter 3.2.4 --- Nucleic acids --- p.87Chapter 3.2.5 --- Kits and reagents --- p.88Chapter 3.2.6 --- Solutions --- p.88Chapter 3.2.7 --- Enzymes --- p.88Chapter 3.2.8 --- Primers --- p.89Chapter 3.3 --- Methods --- p.90Chapter 3.3.1 --- "Construction of pET-P30, pET-GRA2 and pET-ROP2" --- p.90Chapter (a) --- Construction of pET-GRA2 --- p.93Chapter (i) --- Preparation of total RNA from tachyzoite of T. gondii (RH strain) --- p.93Chapter (ii) --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.93Chapter (iii) --- Cloning of pET-GRA2 --- p.94Chapter (b) --- Construction of pET-ROP2 --- p.95Chapter 3.3.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.95Chapter (a) --- "Expression profile of his-tag P30, his-tag ROP2 and his-tag GRA2" --- p.96Chapter (b) --- Western blot of induced bacterial lysate with mono-clonal anti-his-tag antibody --- p.96Chapter (c) --- Western blot of induced bacterial lysate with anti-serum against his-tag MEA --- p.97Chapter (d) --- "Purification of his-tag P30, his-tag GRA2 and his-tag ROP2 from induced bacterial lysate" --- p.97Chapter (e) --- "Western blot of his-tag P30, his-tag GRA2 and his-tag ROP2 with sero- positive serum of mice and human" --- p.98Chapter (f) --- "Western blot of T. gondii lysate by anti-serum against his-tag GRA2, his-tag P30 and his-tag ROP2" --- p.99Chapter 3.3.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.99Chapter (a) --- Isolation of spleenocytes from vaccinated mice --- p.99Chapter (b) --- T-cell proliferation assay --- p.100Chapter (c) --- Determination of Thl/ Th2 cytokines expression profile by RT-PCR --- p.100Chapter 3.3.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.102Chapter 3.4 --- Results --- p.102Chapter 3.4.1 --- Construction of pET-GRA2 and pET-ROP2 --- p.102Chapter 3.4.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.106Chapter 3.4.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.116Chapter 3.4.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.119Chapter 3.5 --- Discussion --- p.121Chapter 3.5.1 --- Construction of pET-GRA2 and pET-ROP2 --- p.121Chapter 3.5.2 --- "Expression and characterization of his-tag P30, his-tag GRA2 and his-tag ROP2" --- p.121Chapter 3.5.3 --- Measurement of cellular immune responses in his-tag MEA vaccinated mice --- p.124Chapter 3.5.4 --- Protection efficacy of different vaccine constructs against lethal challenge --- p.126Chapter Chapter 4 --- : Expression of the multi-epitopic antigen in Arabidopsis thaliana --- p.129Chapter 4.1 --- INTRODUCTION --- p.129Chapter 4.1.1 --- Transgenic plants as vaccine production systems --- p.129Chapter (a) --- Advantages of using plants as bioreactor --- p.130Chapter (b) --- Transgenic plants for vaccine production --- p.131Chapter (c) --- Limitations --- p.135Chapter (i) --- Low yields --- p.135Chapter (ii) --- Plant-specific glycans --- p.135Chapter 4.1.2 --- Model plant - Arabidopsis thaliana --- p.136Chapter 4.1.3 --- Strategies for expressing the transgene (MEA) in plant --- p.137Chapter (a) --- Seed-specific phaseolin promoter --- p.138Chapter (b) --- Lysine-rich protein (LRP) as fusion partner --- p.138Chapter (c) --- Fusion protein with membrane anchor for targeting to specific vacuolar compartments --- p.139Chapter 4.2 --- MATERIALS --- p.141Chapter 4.2.1 --- Bacterial strains --- p.141Chapter 4.2.2 --- Arabidopsis strains --- p.141Chapter 4.2.3 --- Chemicals --- p.141Chapter 4.2.4 --- Antibiotics --- p.141Chapter 4.2.5 --- Nucleic acids --- p.141Chapter 4.2.6 --- Solutions --- p.142Chapter 4.2.7 --- Enzymes and buffers --- p.142Chapter 4.2.8 --- Primers --- p.143Chapter 4.3 --- METHODS --- p.144Chapter 4.3.1 --- Construction of plant expression vectors --- p.144Chapter 4.3.1.1 --- Strategy 1: Lysine-rich protein (LRP) fusion --- p.144Chapter a) --- Construction of pBI121/ Phaseolin Promoter/ LRP1/ MEA/ his-tag/ phaseolin terminator --- p.149Chapter b) --- Construction of pBI121/ Phaseolin Promoter/ LRPIIA/ MEA/ phaseolin terminator --- p.150Chapter 4.3.1.2 --- Strategy 2: Fusion proteins with membrane anchor for vesicle targeting to specific vesicle compartments --- p.151Chapter ai) --- Construction of pBI121/Phaseolin Promoter/ SP/ MEA/ 491/NOS terminator --- p.156Chapter aii) --- Construction of pBI121/Phaseolin Promoter/ SP/ MEA(J)/ 526/ NOS terminator --- p.157Chapter 4.3.1.3 --- Construction of transgenic control pBI121/phaseolin promoter/ MEA/ phaseolin terminator --- p.157Chapter 4.3.2 --- Agrobacterium-mediated transformation of Arabidopsis thaliana by vaccum infiltration --- p.160Chapter (a) --- Preparation of competent cell for Agrobacterium GV3101 --- p.160Chapter (b) --- Transformation of electro-competent agrobacterium with plant expression vector by electroporation --- p.160Chapter (c) --- PCR screening for agrobacterium carrying the desirable plant expression vector --- p.161Chapter (d) --- Vacuum infiltration --- p.161Chapter 4.3.3 --- Screening of homozygous transgenic plants with single insertion of transgene --- p.162Chapter 4.3.4 --- Detection of MEA or MEA fusion protein in transgenic plants --- p.163Chapter (a) --- Extraction of seed protein (Soluble and membrane fractions) --- p.163Chapter (b) --- Western blot of seed protein by anti-serum against HisMEA --- p.163Chapter 4.4 --- RESULTS --- p.164Chapter 4.4.1 --- Construction of plant expression vectors and transgenic A. thaliana --- p.164Chapter (a) --- Construction of pBI121/ phaseolin promoter/ LRPI or LRP II/ MEA/ Phaseolin terminator --- p.164Chapter (b) --- Construction of pBI121/ PP/ SP/ MEA(J)/ 491 or 526/ PT --- p.165Chapter (c) --- Construction of transgenic control pBI121/ PP/ MEA/ PT --- p.166Chapter (d) --- Agrobacterium-mediated transformation of A. thaliana --- p.167Chapter 4.4.2 --- Screening of homozygous transgenic plant with single insertion of transgene --- p.172Chapter 4.4.3 --- Molecular analysis of the MEA/ MEA fusion in transgenic plants --- p.181Chapter 4.5 --- DISCUSSION --- p.188Chapter 4.5.1 --- Construction of plant expression vector --- p.188Chapter 4.5.2 --- Screening of homozygous transgenic plant with single insertion of transgene --- p.189Chapter 4.5.3 --- Molecular analysis of transgenic MEA/ MEA fusion in transgenic plants --- p.190Chapter Chapter 5 --- : General Discussion --- p.193Chapter 5.1 --- Development of a vaccine for toxoplasmosis: current status --- p.193Chapter 5.1.1 --- Current status --- p.193Chapter (a) --- Vaccine for agricultural animals --- p.193Chapter (b) --- Other vaccine candidates and the protective response induced --- p.194Chapter 5.1.2 --- Appropriate vaccine design - the multi-epitopic antigen (MEA) --- p.194Chapter 5.2 --- "Expression, Characterization and Immunological studies of the MEA expressed in prokaryotic system" --- p.195Chapter 5.2.1 --- Expression and characterization of E. coli- expressed his-tag MEA --- p.195Chapter 5.2.2 --- Immunological studies on the E. coli- expressed his-tag MEA --- p.196Chapter 5.3 --- Expression of MEA in transgenic plants --- p.197Chapter 5.4 --- Preclinical Safety Assessment: Considerations in Vaccine Development --- p.198References --- p.20