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