Law Pui-ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 98-105). / Abstracts in English and Chinese. / Statement --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / Table of contents --- p.viii / List of figures --- p.xv / List of tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Schistosomiasis --- p.1 / Chapter 1.1.1 --- Disease burden --- p.1 / Chapter 1.1.2 --- Causative agents --- p.1 / Chapter 1.1.3 --- Transmission --- p.2 / Chapter 1.1.4 --- Pathology of the disease --- p.3 / Chapter 1.1.5 --- Control and therapy --- p.3 / Chapter 1.2 --- Schistosomiasis in China --- p.5 / Chapter 1.3 --- Schistosoma japonicum --- p.6 / Chapter 1.3.1 --- Life cycle of S. japonicum --- p.6 / Chapter 1.3.2 --- Biology of S. japonicum cercaria --- p.9 / Chapter 1.3.3 --- Transformation of cercaria to schistosomulum --- p.10 / Chapter 1.3.4 --- Cercarial stage-specific antigens --- p.12 / Chapter 1.4 --- Aim of study --- p.14 / Chapter Chapter 2 --- Materials and methodology --- p.15 / Chapter 2.1 --- Materials --- p.15 / Chapter 2.1.1 --- Schistosoma japonicum cercaria cDNA library --- p.15 / Chapter 2.1.2 --- Cercarial stage-specific clones --- p.15 / Chapter 2.1.3 --- Adult worm and cercaria RNA --- p.18 / Chapter 2.1.4 --- "Snail intermediate host, Oncomelania hupensis" --- p.18 / Chapter 2.1.5 --- Bacterial strains --- p.18 / Chapter 2.1.6 --- Chemicals --- p.19 / Chapter 2.1.7 --- Kits and reagents --- p.21 / Chapter 2.1.8 --- Nucleic acids --- p.22 / Chapter 2.1.9 --- Solutions --- p.22 / Chapter 2.1.10 --- Enzymes --- p.24 / Chapter 2.1.11 --- Primers --- p.25 / Chapter 2.1.12 --- Antibodies --- p.26 / Chapter 2.2 --- Methodology --- p.27 / Chapter 2.2.1 --- Identification of cercarial stage-specific genes --- p.27 / Chapter 2.2.1.1 --- Sequence analysis of cercarial stage-specific clones --- p.27 / Chapter 2.2.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.29 / Chapter 2.2.1.2.1 --- Cloning of S. japonicum glyceraldehyde-3-phosphate dehydrogenase (sjGAPDH) (GenBank accession no. U75571) --- p.29 / Chapter 2.2.1.2.1.1 --- Reverse transcription and PCR amplification of sjGAPDH --- p.29 / Chapter 2.2.1.2.1.2 --- Cloning of sjGADPH in pBluescript II KS (-) --- p.29 / Chapter 2.2.1.2.1.3 --- Sequence verification of cloned sjGAPDH --- p.30 / Chapter 2.2.1.2.2 --- Synthesis of Digoxigenin (DIG)-labeled probe --- p.31 / Chapter 2.2.1.2.2.1 --- Synthesis of DIG-labeled probe by PCR --- p.31 / Chapter 2.2.1.2.2.2 --- Estimation of concentration of DIG-labeled probe --- p.32 / Chapter 2.2.1.2.3 --- RNA dot blot --- p.33 / Chapter 2.2.1.2.3.1 --- Transferring RNA to the membrane using the BIO-RAD blotting manifold --- p.33 / Chapter 2.2.1.2.3.2 --- Hybridization of DIG-labeled probe to detect sjGAPDH --- p.33 / Chapter 2.2.1.2.3.3 --- Detection of the chemiluminescent signal --- p.33 / Chapter 2.2.1.2.3.4 --- Stripping membrane for reprobing --- p.34 / Chapter 2.2.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.35 / Chapter 2.2.2.1 --- Cloning of full-length cDNA of 20H8 --- p.35 / Chapter 2.2.2.1.1 --- 5'RACE of 20H8 --- p.35 / Chapter 2.2.2.1.2 --- Cloning of full-length 20H8 into pBluescript II SK(-) --- p.36 / Chapter 2.2.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.37 / Chapter 2.2.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.38 / Chapter 2.2.2.3.1 --- Expression of 20H8 in E. coli --- p.38 / Chapter 2.2.2.3.1.1 --- "Cloning of 20H8 in an E coli expression vector, pET32a+" --- p.38 / Chapter 2.2.2.3.1.2 --- Expression of recombinant 20H8 protein in E. coli --- p.39 / Chapter 2.2.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.40 / Chapter 2.2.2.3.3 --- Production of antiserum --- p.40 / Chapter 2.2.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.41 / Chapter 2.2.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.42 / Chapter 2.2.2.4 --- Immunolocalization of 20H8 in cercaria --- p.43 / Chapter 2.2.2.4.1 --- Collection of S. japonicum cercaria --- p.43 / Chapter 2.2.2.4.2 --- Immunofluorescence staining of cercaria --- p.43 / Chapter 2.2.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.45 / Chapter 2.2.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.45 / Chapter 2.2.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.46 / Chapter 2.2.3.2.1 --- Expression of sjCa8 in E. coli --- p.46 / Chapter 2.2.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.46 / Chapter 2.2.3.2.3 --- Production of antiserum --- p.46 / Chapter 2.2.3.2.4 --- Evaluation of the immunogenicity of sjCa8 --- p.47 / Chapter 2.2.3.2.5 --- Evaluation of the antigenicity of sjCa8 --- p.47 / Chapter 2.2.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.48 / Chapter 2.2.3.3.1 --- Calcium-dependent electrophoretic mobility shift --- p.48 / Chapter 2.2.3.3.2 --- Ruthenium red assay --- p.48 / Chapter 2.2.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.49 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1. --- Identification of cercarial stage-specific genes --- p.50 / Chapter 3.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.50 / Chapter 3.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.54 / Chapter 3.2. --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.57 / Chapter 3.2.1 --- Cloning of full-length cDNA of 20H8 --- p.57 / Chapter 3.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.59 / Chapter 3.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.62 / Chapter 3.2.3.1 --- Expression of 20H8 in E. coli --- p.62 / Chapter 3.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.64 / Chapter 3.2.3.3 --- Production of antiserum --- p.65 / Chapter 3.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.66 / Chapter 3.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.67 / Chapter 3.2.4 --- Immunolocalization of 20H8 in cercaria --- p.68 / Chapter 3.3. --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.70 / Chapter 3.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.70 / Chapter 3.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.75 / Chapter 3.3.2.1 --- Expression of sjCa8 in E. coli --- p.75 / Chapter 3.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.76 / Chapter 3.3.2.3 --- Production of anti-sjCa8 serum --- p.77 / Chapter 3.3.2.4 --- Evaluation of immunogenicity of sjCa8 protein --- p.77 / Chapter 3.3.2.5 --- Evaluation of antigenicity of sjCa8 protein --- p.78 / Chapter 3.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.79 / Chapter 3.3.3.1 --- Electrophoretic motility shift --- p.80 / Chapter 3.3.3.2 --- Ruthenium red binding assay --- p.80 / Chapter 3.3.4 --- Immunolocalization of sjCa8 in cercaria 81 --- p.81 / Chapter Chapter 4 --- Discussion --- p.83 / Chapter 4.1 --- Identification of cercarial stage-specific genes --- p.83 / Chapter 4.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.83 / Chapter 4.1.2 --- "Confirmation of stage-specific expression of the selected genes, 20H8 and sjCa8" --- p.85 / Chapter 4.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.86 / Chapter 4.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.86 / Chapter 4.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.88 / Chapter 4.2.4 --- Immunolocalization of 20H8 in cercaria --- p.90 / Chapter 4.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.91 / Chapter 4.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.91 / Chapter 4.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.93 / Chapter 4.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.94 / Chapter 4.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.94 / Chapter 4.4 --- Conclusions --- p.95 / References --- p.96

Lok Chui-Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgement --- p.II / Abstract --- p.IV / Chinese Abstract (摘要） --- p.VI / Abbreviation --- p.VIII / Table of Contents --- p.XIII / List of Tables --- p.XVII / List of Figures --- p.XVIII / Chapter Chapter One : --- Literature Review --- p.1 / Chapter 1.1 --- The Schistosoma Species --- p.1 / Chapter 1.1.1 --- The Schistosoma Gene Discovery --- p.3 / Chapter 1.1.2 --- Schistosome Transcriptome --- p.4 / Chapter 1.2 --- Schistosomiasis --- p.4 / Chapter 1.2.1 --- Immunopathology of Schistosomiasis --- p.5 / Chapter 1.2.2 --- Diagnosis of Schistosomiasis --- p.7 / Chapter 1.2.3 --- Treatment and Control for Schistosomiasis --- p.7 / Chapter 1.2.4 --- Vaccine Development for Schistosomiasis --- p.8 / Chapter 1.3 --- "The Species, Schistosoma japonicum" --- p.9 / Chapter 1.3.1 --- The Life Cycle of Schistosoma japonicum --- p.10 / Chapter 1.3.1.1 --- "The Egg, Miracidium Phase of the Life Cycle" --- p.12 / Chapter 1.3.1.2 --- Developmental Cycle within Mollusc Host --- p.12 / Chapter 1.3.1.3 --- The Cercaria Phase of Life Cycle --- p.13 / Chapter 1.3.1.4 --- Adult Schistosome in Definitive Host --- p.14 / Chapter 1.4 --- Invasion by Schistosome Cercariae --- p.15 / Chapter 1.5 --- "The Anti-inflammatory Protein, Sml6" --- p.16 / Chapter 1.5.1 --- Discovery of Sm 16 --- p.16 / Chapter 1.5.2 --- Cloning and Expression of Gene-encoding Sm 16 --- p.17 / Chapter 1.5.3 --- Potential Anti-inflammatory Therapy using Sm 16 --- p.18 / Chapter 1.6 --- Innate Immunity and Adaptive Immunity --- p.18 / Chapter 1.6.1 --- Macrophage --- p.18 / Chapter 1.6.2 --- Major Histocompatiblity Complex (MHC) --- p.20 / Chapter 1.6.3 --- Adaptive Immunity to Parasites --- p.20 / Chapter 1.7 --- Inflammation --- p.21 / Chapter 1.7.1 --- Cells of the Inflammatory Process --- p.23 / Chapter 1.7.2 --- Cytokines --- p.24 / Chapter 1.7.2.1 --- Interleukin-1 (IL-1) System --- p.26 / Chapter 1.7.2.2 --- Interferon (IFN) System --- p.27 / Chapter 1.7.3 --- Anti-inflammatory Therapy --- p.28 / Chapter 1.8 --- Aim of Study --- p.29 / Chapter Chapter Two : --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Cell Lines, Mouse Strain and Bacterial Strains" --- p.30 / Chapter 2.1.2 --- Plasmids --- p.31 / Chapter 2.1.3 --- Chemicals --- p.31 / Chapter 2.1.4 --- "Kits, Nucleic Acids and Reagents" --- p.34 / Chapter 2.1.5 --- Antibodies and Immunoglobins --- p.35 / Chapter 2.1.6 --- Cell Culture Reagents --- p.35 / Chapter 2.1.7 --- Solutions --- p.36 / Chapter 2.1.8 --- Solutions of Reaction Kits --- p.39 / Chapter 2.1.9 --- Enzymes --- p.41 / Chapter 2.1.10 --- Major Equipments and Materials --- p.41 / Chapter 2.1.11 --- Primers --- p.43 / Chapter 2.1.11.1 --- Sequencing and Sj 16 Gene-coding Specific Primers --- p.43 / Chapter 2.1.11.2 --- Primers for Cytokines --- p.43 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Amplification of Sjl6 cDNA from Schistosoma japonicum Cercariae --- p.45 / Chapter 2.2.1.1 --- Isolation of Cercariae total RNA by Guanidinium Thiocyanate - Cesium Chloride Ultracentrifugation --- p.45 / Chapter 2.2.1.2 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 2.2.1.2.1 --- Reverse Transcription (RT) --- p.46 / Chapter 2.2.1.2.2 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.2 --- Cloning and Subcloning of Sj 16 --- p.47 / Chapter 2.2.2.1 --- Preparation of DH5a Competent Cells --- p.47 / Chapter 2.2.2.2 --- Purification of Plasmid DNA --- p.48 / Chapter 2.2.2.3 --- Restriction Enzyme Digestion of DNA --- p.49 / Chapter 2.2.2.4 --- Purification of DNA Fragments from Agarose Gel --- p.50 / Chapter 2.2.2.5 --- Ligation of Purified DNA Fragments --- p.51 / Chapter 2.2.2.6 --- Transformation of Recombinant Plasmid --- p.52 / Chapter 2.2.2.7 --- Selection of Transformed Clones --- p.52 / Chapter 2.2.2.7.1 --- Screening by X-gal and IPTG : a-complementation --- p.52 / Chapter 2.2.2.7.2 --- Screening by Polymerase Chain Reaction --- p.53 / Chapter 2.2.2.8 --- Cycle Sequencing --- p.53 / Chapter 2.2.3 --- Expression of the rSj 16 in Eukaryotic System --- p.55 / Chapter 2.2.3.1 --- Transfection of pSecTag2B/Sj 16 Plasmid into Animal Cells --- p.55 / Chapter 2.2.3.2 --- PCR Screening of Transfected Cells --- p.56 / Chapter 2.2.3.3 --- Analysis of mRNA Transcript by RT-PCR --- p.56 / Chapter 2.2.3.4 --- Concentration of the Condition Medium --- p.57 / Chapter 2.2.3.5 --- Western Blot analysis of rSjl6 Expression --- p.58 / Chapter 2.2.4 --- Expression of rSjl6 in Bacterial System --- p.59 / Chapter 2.2.4.1 --- Transformation of pET30a+/Sjl6 Plasmid into BL21 --- p.59 / Chapter 2.2.4.2 --- Optimization of rSj 16 Expression --- p.60 / Chapter 2.2.4.3 --- Solubility of the rSjl6 --- p.60 / Chapter 2.2.4.4 --- Estimation of rSj 16 Concentration --- p.62 / Chapter 2.2.4.5 --- Western Blot Analysis of rSj 16 --- p.62 / Chapter 2.2.5 --- Recombinant Protein Purification --- p.63 / Chapter 2.2.5.1 --- Affinity Chromatography of Recombinant Protein --- p.63 / Chapter 2.2.5.2 --- Dialysis of Eluted Recombinant Protein in PBS --- p.64 / Chapter 2.2.5.3 --- Estimation of Recombinant Protein Concentration --- p.65 / Chapter 2.2.6 --- Demonstrate the Anti-inflammatory Activity of rSj 16 --- p.65 / Chapter 2.2.6.1 --- Thioglycollate Induced Macrophage Recruitment --- p.65 / Chapter 2.2.6.2 --- Cytospin and Hemacolor Staining of PECs --- p.66 / Chapter 2.2.6.3 --- FACS Analysis of PECs --- p.67 / Chapter 2.2.6.4 --- Isolation of total RNA by TRIZOL Reagent --- p.67 / Chapter 2.2.7 --- Immunogenicity and Antigenicity of rSjl6 --- p.68 / Chapter 2.2.7.1 --- Western Blot of rSjl6 with Schistosoma japonicum infected rabbit serum --- p.69 / Chapter 2.2.7.2 --- Preparation of Anti-Sj 16 Serum --- p.69 / Chapter 2.2.7.3 --- Western Blot of rSjl6 with immunized mice serum --- p.70 / Chapter 2.2.8 --- FACS analysis of MHC (I) Expression --- p.71 / Chapter 2.2.9 --- Anti-proliferative Assay using BrdU Kit --- p.72 / Chapter Chapter Three : --- Results --- p.73 / Chapter 3.1 --- Amplification of Sj 16 cDNA from Schistosoma japonicum Cercariae total RNA --- p.73 / Chapter 3.2 --- Construction of pBluescript II SK(-) / Sjl6 --- p.75 / Chapter 3.3 --- Analysis of Sj 16 Nucleotide and Amino Acid Sequence --- p.78 / Chapter 3.3.1 --- Blastn Search Analysis --- p.80 / Chapter 3.3.2 --- Blastx Search Analysis --- p.82 / Chapter 3.3.3 --- Structural Analysis --- p.84 / Chapter 3.4 --- Subcloning of Sjl6 cDNA into pET30a+ and pSecTag2B Expression Vector --- p.88 / Chapter 3.5 --- Expression of the rSj 16 --- p.92 / Chapter 3.5.1 --- Animal Cell Expression --- p.92 / Chapter 3.5.1.1 --- Analysis of mRNA Transcript by RT-PCR --- p.93 / Chapter 3.5.1.2 --- Western Blot of Condition Medium --- p.95 / Chapter 3.5.2 --- Bacterial Cell Expression --- p.97 / Chapter 3.5.2.1 --- Optimization of rSjl6 Expression --- p.97 / Chapter 3.5.2.2 --- Estimation of rSjl6 Concentration --- p.98 / Chapter 3.5.2.3 --- Solubility of rSj16 --- p.99 / Chapter 3.5.2.4 --- Western Blot Analysis of rSjl6 --- p.100 / Chapter 3.6 --- Purification of Recombinant Protein --- p.101 / Chapter 3.6.1 --- Purification of rSj16 --- p.101 / Chapter 3.6.2 --- Purification of rSjCa8 --- p.104 / Chapter 3.7 --- Anti-inflammatory Activity of rSj 16 --- p.107 / Chapter 3.7.1 --- Analysis of PECs in Thioglycollate Induced Inflammation --- p.107 / Chapter 3.7.2 --- Hemacolor Staining of PECs --- p.110 / Chapter 3.7.3 --- FACS Analysis of PECs --- p.110 / Chapter 3.7.4 --- RT-PCR of RNA Isolated from PECs --- p.115 / Chapter 3.8 --- Immunogenicity and Antigenicity of rSjl6 --- p.117 / Chapter 3.8.1 --- Immunogenicity of rSj 16 --- p.117 / Chapter 3.8.2 --- Antigenicity of rSj16 --- p.117 / Chapter 3.9 --- Inhibitory Effect of rSj 16 on rMuIFN-a4 Induced Up-regulation of MHC(I) Expression --- p.120 / Chapter 3.9.1 --- Time Course of rMuIFN-α4 Induced Up-regulation of MHC(I) Expression --- p.120 / Chapter 3.9.2 --- Inhibitory Effect of rSjl6 on rMuIFN-α4 Induced MHC (I) Up-regulation --- p.120 / Chapter 3.9.3 --- "Anti-proliferation Effect of rMuIFN-a4, rSj 16 and rSjCa 8" --- p.124 / Chapter 3.9.4 --- Effect of Signal Transduction Inhibitors on rMuIFN-a4 Induced MHC (I) Up-regulation --- p.126 / Chapter Chapter Four : --- Discussion and Conclusion --- p.129 / Chapter 4.1 --- Discussion --- p.129 / Chapter 4.1.1 --- Overview --- p.129 / Chapter 4.1.2 --- Molecular and Structural Analysis of rSj 16 --- p.130 / Chapter 4.1.3 --- Relationship between Sml6 and Sjl6 --- p.131 / Chapter 4.1.4 --- Anti-inflammatory Activity of rSj 16 --- p.132 / Chapter 4.1.5 --- Immunogenicity and Antigenicity of rSjl6 --- p.137 / Chapter 4.1.6 --- Inhibitory Effect of rSjl6 on rMuIFN-a4 Induced Up-regulation of MHC (I) Expression --- p.138 / Chapter 4.1.7 --- Relation between Sj 16 and the Innate Immune System --- p.139 / Chapter 4.1.8 --- Further Study and Significance --- p.140 / Chapter 4.2 --- Conclusion --- p.141 / References --- p.142