Li Chi Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 137-162). / Abstracts in English and Chinese. / Statement --- p.iii / Acknowledgments --- p.iv / Abstract --- p.vi / Abstract (Chinese version) --- p.viii / Abbreviations --- p.x / Table of contents --- p.xii / List of Figure --- p.xviii / List of Table --- p.xx / Chapter Chapter One --- Literature Review / Chapter 1.1 --- The Genus Schistosoma --- p.1 / Chapter 1.2 --- Biology of Schistosoma japonicum --- p.3 / Chapter 1.2.1 --- The History of discovery of Schitosoma japonicum --- p.3 / Chapter 1.2.2 --- Life cycle of Schistosoma japonicum --- p.3 / Chapter 1.2.2.1 --- Egg --- p.6 / Chapter 1.2.2.2 --- Miracidia --- p.6 / Chapter 1.2.2.3 --- Sporocysts --- p.7 / Chapter 1.2.2.4 --- Cercaria --- p.9 / Chapter 1.2.2.5 --- Schislosomula --- p.10 / Chapter 1.2.2.6 --- Adult worms --- p.10 / Chapter 1.2.3 --- Genetics of Schistosoma japonicum --- p.11 / Chapter 1.2.3.1 --- Genome analysis --- p.12 / Chapter 1.2.3.2 --- Schistosome genome --- p.13 / Chapter 1.2.4 --- Tegumental membrane of Schistosomes --- p.14 / Chapter 1.3 --- Pathology of Schistosomiasis --- p.15 / Chapter 1.3.1 --- Acute Schistosomiasis --- p.15 / Chapter 1.3.2 --- Intestinal Disease --- p.16 / Chapter 1.3.3 --- Hepatosplenic Disease --- p.16 / Chapter 1.3.4 --- Cerebral Schistosomiasis --- p.16 / Chapter 1.4 --- Treatment of Schistosomiasis --- p.17 / Chapter 1.4.1 --- Chemotherapy --- p.17 / Chapter 1.4.2 --- Schistosoma Vaccine --- p.17 / Chapter 1.5 --- GPI anchor --- p.19 / Chapter 1.5.1 --- Function of GPI anchored proteins --- p.19 / Chapter 1.5.2 --- Synthesis of GPI anchor --- p.21 / Chapter 1.5.3 --- "Phosphatidylinositol Glycan, Class N (PIG-N)" --- p.26 / Chapter 1.6 --- The role of GPI anchor proteins in Schistosome --- p.26 / Chapter 1.7 --- Aim of study --- p.29 / Chapter Chapter two --- Materials and Methods / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Cell lines and Bacterial Strains --- p.31 / Chapter 2.1.2 --- Chemicals --- p.32 / Chapter 2.1.3 --- Kits and Reagents --- p.34 / Chapter 2.1.4 --- Nucleic acids --- p.34 / Chapter 2.1.5 --- Reagents for Cell culture --- p.34 / Chapter 2.1.6 --- Solutions --- p.35 / Chapter 2.1.7 --- Enzymes --- p.37 / Chapter 2.1.8 --- Primer List --- p.37 / Chapter 2.1.9 --- Antibodies --- p.39 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Screening of the S. japonicum cercaria stage cDNA library --- p.40 / Chapter 2.2.1.1 --- λ phage plating --- p.40 / Chapter 2.2.1.2 --- Single plaque isolation --- p.40 / Chapter 2.2.1.3 --- Conversion of Lambda TriplEx to pTriplEx --- p.41 / Chapter 2.2.1.4 --- preparation of plasmid DNA --- p.41 / Chapter 2.2.1.5 --- cycle DNA sequencing --- p.42 / Chapter 2.2.2. --- RT-PCR --- p.44 / Chapter 2.2.2.1 --- Isolation of Total RNA by Guandidium Thiocyanate - Cesium Chloride ultracentrifugation --- p.44 / Chapter 2.2.2.2 --- Synthesis of Fist Strand cDNA by reverse transcriptation reaction --- p.45 / Chapter 2.2.2.3 --- PGR amplification of RT product --- p.46 / Chapter 2.2.3 --- Rapid Amplification of cDNA Ends (RACE) --- p.46 / Chapter 2.2.3.1 --- Synthesis of first strand cDNA for RACE reaction --- p.46 / Chapter 2.2.3.2 --- 5 ´ةRACE for Sj-PIG-N gene --- p.47 / Chapter 2.2.3.3 --- 3´ة RACE for Sj-PIG-N gene --- p.48 / Chapter 2.2.3.4 --- Purification of DNA fragment from agarose gel --- p.48 / Chapter 2.2.3.5 --- Ligation of purified PCR fragments and pBluescriptII KS(+) T-vector --- p.49 / Chapter 2.2.3.6 --- Preparation of DH5a competent cells --- p.49 / Chapter 2.2.3.7 --- Transformation of recombinant plasmid --- p.50 / Chapter 2.2.4 --- Mammalian cell transfection --- p.50 / Chapter 2.2.4.1 --- Stable transfection --- p.50 / Chapter 2.2.4.2 --- Transient transfection --- p.51 / Chapter 2.2.5 --- Biological function studies of Sj-PIG-N gene --- p.52 / Chapter 2.2.5.1 --- Flow Cytometry (FCM) analysis --- p.52 / Chapter 2.2.5.2 --- In Vitro Mannose Labeling of Microsomes and Characterization of Glycolipids --- p.53 / Chapter 2.2.6 --- Intracellular localization assay --- p.54 / Chapter Chapter Three --- Results / Chapter 3.1 --- Random sequence analysis of cercaria EST from S. japonicum Cercaria stage cDNA library --- p.55 / Chapter 3.1.1 --- Sequencing results --- p.57 / Chapter 3.1.2 --- BlastX Search results --- p.61 / Chapter 3.2 --- The expression of Sj-PIG-N gene in both adult worms and cercaria --- p.68 / Chapter 3.2.1 --- Spectrophotometric analysis of total RNA --- p.68 / Chapter 3.2.2 --- Detection of Sj-PIG-N gene expression in both adult worm and cercaria stages --- p.70 / Chapter 3.3 --- Cloning of the full length of Sj-PIG-N gene --- p.72 / Chapter 3.3.1 --- Amplification of the full length of Sj-PIG-N gene --- p.72 / Chapter 3.3.2 --- Obtaining the full length sequence of Sj-PIG-N gene --- p.74 / Chapter 3.4 --- Sequence analysis of Ml length Sj-PIG-N cDNA --- p.80 / Chapter 3.5 --- Analysis of Sj-PIG-N protein structure by computer programs --- p.91 / Chapter 3.6 --- Construction of Sj-PIG-N gene into mammalian cell expression vector pEGFP-Nl and pEGFP-Hyg --- p.97 / Chapter 3.6.1 --- Construction of pBluescriptII KS(+)-Sj-PIG-N-B --- p.97 / Chapter 3.6.2 --- Construction of pBluescriptll KS(+)-Sj-PIG-N-E --- p.101 / Chapter 3.6.3 --- Construction of pTRE2-Sj-PIG-N --- p.106 / Chapter 3.6.4 --- Construction of pEGFP-Hyg-Sj-PIG-N --- p.109 / Chapter 3.6.4.1 --- Construction of pEGFP-Hyg --- p.109 / Chapter 3.6.4.2 --- Construction of pEGFP-Hyg-Sj-PIG-N --- p.114 / Chapter 3.7 --- Molecular analysis of Sj-PIG-N gene --- p.117 / Chapter 3.7.1 --- Functional analysis of Sj-PIG-N --- p.117 / Chapter 3.7.1.1 --- FACS analysis of surface expression of GPI-anchored protein - Thyl --- p.117 / Chapter 3.7.1.2 --- FACS analysis of surface expression of GPI-anchor protein - CD24 --- p.119 / Chapter 3.7.1.3 --- In Vitro Mannose Labeling of Microsomes and Characterization of Glycolipids --- p.121 / Chapter 3.7.2 --- Localization of Sj-PIG-N with immunofluorescent staining --- p.123 / Chapter Chapter Four --- Discussion / Chapter 4.1 --- S. japonicum cercaria EST analysis --- p.125 / Chapter 4.2 --- Structure analysis of Sj-Pig-N gene --- p.128 / Chapter 4.3 --- Molecular analysis of Sj-PIG-N --- p.131 / Chapter 4.4 --- Further study --- p.134 / Chapter 4.5 --- Conclusion --- p.136 / References --- p.137
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324590 |
| Date | January 2004 |
| Contributors | Li, Chi Ho., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxi, 162 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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