Characterization of Sj16 in Schistosoma japonicum.

• Other Authors: Lok, Chui-Lin.
• Format: Others
• Language: English; Chinese
• Published: 2005
• Subjects: Schistosoma japonicum; Anti-inflammatory agents; Schistosomiasis; Schistosoma japonicum > genetics; Anti-Inflammatory Agents; Schistosomiasis > immunology
• Online Access: http://library.cuhk.edu.hk/record=b5892543; http://repository.lib.cuhk.edu.hk/en/item/cuhk-325364

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