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

Schistosome is the causative agent of schistosomiasis which is one of the world's most prevalent tropical diseases. In the skin of infected host, significant inflammatory response to the parasite is not observed. Previous studies from Schistosoma mansoni showed that this subdued inflammatory response was due to a 16-KDa protein, Sm16, which is present abundantly in the secretions of schistosomulae. Provided that Schistosoma japonicum shares the same infective pathway as S. mansoni by penetrating the skin, it seems logical that S. japonicum has a protein with a similar role to Sm16 to down-regulate host immune responses. According to the cDNA sequence of Sm16, a corresponding gene (designated Sj16) of Sm16 has previously been amplified and cloned from the cercarial cDNA of S. japonicum. Sequence analysis showed that Sj16 shares 99% identity with Sm16 in its nucleotide sequence, and 100% identity in its protein sequence. While previous studiers reported their failure in obtaining the soluble recombinant protein of Sm16, we expressed and purified the recombinant Sj16 (rSj16) from E. coli in the present study. Western blot and ELISA analysis showed that S. japonicum-infected rabbit sera could not recognize rSj16, indicating that native Sj16 might fail to induce circulating antibodies during S. japonicum infection. In the in vivo study, rSj16 dramatically suppressed not only the recruitment of leukocytes to the peritoneal cavity of BALB/c mice injected with thioglycollate, but also the maturation of thioglycollate-induced peritoneal macrophages. The suppression effect was accompanied by a marked up-regulation of IL-10 and IL-1RA transcripts, and down-regulation of IL-12p35, IL-1beta and MIP-2 transcripts in peritoneal cells. Further analysis revealed that rSj16 also inhibited both humoral and cellular immune responses to heterologous antigens. In addition, rSj16 was found to induce macrophage differentiation of the murine myeloid leukemia WEHI-3B (JCS) cells, and regulate the differentiation of mouse hematopoietic cells towards the macrophage lineage. Although previous studies indicated the involvement of endogenous IL-1alpha, IL-1beta and TNF-alpha in the macrophage differentiation of JCS cells, the results from this study suggested that rSj16-induced JCS cell differentiation do not rely on the endogenous production of these three cytokines. This is the first study to successfully express and purify sufficient soluble rSj16, and demonstrate the anti-inflammatory and immunomodulatory effects of the rSj16. / Hu, Shaomin. / Adviser: Ming Chiu Fung. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0210. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 139-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.