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Molecular analysis of schistosoma japonicum phosphatidylinositol glycan -- class N gene.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324590
Date January 2004
ContributorsLi, Chi Ho., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xxi, 162 leaves : ill. (some col.) ; 30 cm.
RightsUse 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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