Kit Yu Fu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 99-106). / Chapter I. --- Abstract --- p.vii / Chapter II. --- Acknowledgements --- p.viii / Chapter III. --- List of Figures --- p.ix / Chapter IV. --- Chapter --- p.1 / Introduction --- p.1 / Chapter 1.1 --- Laboratory diagnosis of infectious diseases --- p.1 / Chapter a. --- Culture --- p.1 / Chapter b. --- Direct detection by visualization --- p.2 / Chapter c. --- Direct detection by DNA or RNA hybridization --- p.2 / Chapter d. --- Detection by immunological methods (antigen or antibody detection) --- p.3 / Chapter 1.2 --- Types of antigen preparations / Chapter a. --- Crude antigenic extracts --- p.5 / Chapter b. --- Affinity-purified antigens --- p.6 / Chapter c. --- Recombinant antigens --- p.6 / Chapter 1.3 --- Methods of gene transfer to mammalian cells --- p.8 / Chapter 1.4 --- The immunoglobulins --- p.10 / Chapter 1.4.1 --- Ig structure --- p.11 / Chapter 1.4.2 --- Ig genes --- p.13 / Chapter 1.4.3 --- Ig gene rearrangement --- p.15 / Chapter 1.4.4 --- Recombinant Ig --- p.15 / Chapter 1.4.5 --- Myeloma-derived recombinant Ig (chimeric antibodies) --- p.17 / Chapter 1.4.6 --- Ig expression vectors --- p.19 / Chapter 1.5 --- Trichinella spiralis and trichinosis --- p.20 / Chapter 1.5.1 --- The parasite --- p.21 / Chapter 1.5.2 --- Antigens of T. spiralis --- p.21 / Chapter 1.6 --- Aim of present study --- p.25 / Chapter V. --- Chapter2 / Materials and Methods / Chapter 2.1 --- Chemicals --- p.27 / Chapter 2.2 --- Parasite --- p.27 / Chapter 2.3 --- Cell line and expression vectors --- p.28 / Chapter 2.4 --- Extraction of total RNA from T. spiralis --- p.29 / Chapter 2.5 --- Preparation of cDNA fragment from T. spiralis --- p.29 / Chapter 2.6 --- Characterization of Trichinella cDNA fragment --- p.31 / Chapter 2.6.1 --- By gel electrophoresis --- p.31 / Chapter 2.6.2 --- By restriction enzyme digestion --- p.31 / Chapter 2.7 --- Cloning of Trichinella cDNA fragment to g4R --- p.31 / Chapter 2.7.1 --- Preparation Trichinella cDNA fragment for ligation --- p.32 / Chapter 2.7.2 --- Preparation of g4R vector --- p.32 / Chapter 2.7.3 --- Ligation --- p.32 / Chapter 2.7.4 --- Transformation of Escherichia coli (TG1) / Chapter a. --- Preparation of competent cells for transformation --- p.33 / Chapter b. --- Transformation of competent cells by heat shock --- p.34 / Chapter 2.7.5 --- Screening of recombinant clones --- p.34 / Chapter 2.8 --- Preparation of fusion gene for transfection --- p.36 / Chapter 2.9 --- Introduction of DNA to myeloma cells by electroporation --- p.36 / Chapter 2.10 --- Enzyme-linked immunosorbent assay (ELISA) to detect fusion gene product / Chapter 2.10.1 --- Sandwich ELISA --- p.37 / Chapter 2.10.2 --- Detection of Trichinella antigen in fusion gene product --- p.38 / Chapter 2.10.3 --- Detection of Ig CH2-CH3 domains in fusion gene product --- p.38 / Chapter 2.11 --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.39 / Chapter 2.12 --- Genomic and transcriptional analysis of transfectants --- p.40 / Chapter 2.12.1 --- Genomic analysis of transfectants / Chapter a. --- DNA isolation --- p.40 / Chapter b. --- PCR amplification of the fusion gene fragment --- p.41 / Chapter 2.12.2 --- Isolation of fusion gene cDNA from transfectants --- p.41 / Chapter 2.12.3 --- Cloning of fusion gene cDNA to M13 mpl9 --- p.43 / Chapter 2.12.4 --- Preparation of single-stranded templates from M13 phage --- p.43 / Chapter 2.12.5 --- Dideoxy sequencing method (Sanger) --- p.44 / Chapter 2.12.6 --- Gel analysis of sequencing products --- p.44 / Chapter 2.13 --- Modification of the g4R expression vector by deletion of the CH2-CH3 exons 3' to the XhoI site / Chapter a. --- Partial EcoRI digestion of g4R --- p.45 / Chapter b. --- Addition of adaptors to the partial Eco RI-digested g4R vector --- p.46 / Chapter c. --- Preparation of modified g4R vector --- p.46 / Chapter 2.14 --- Cloning of the Trichinella P53 gene into the modified g4R vector --- p.46 / Chapter 2.15 --- Detection of Trichinella antigen in the second fusion gene product / Chapter a. --- Preparation of biotinylated mouse anti- Trichinella serum --- p.47 / Chapter b. --- Assay for the activity of biotin-Ts serum --- p.48 / Chapter c. --- "Assay for detection of Trichinella antigen in the fusion gene product from Tc2, Te1 and g4R transfected clones" --- p.48 / Chapter 2.16 --- Northern blot analysis of the RNA of transfected clones --- p.48 / Chapter a. --- RNA gel electrophoresis --- p.49 / Chapter b. --- RNA transfer --- p.49 / Chapter c. --- RNA hybridization --- p.49 / Chapter VI. --- "Chapter3 Construction of Ig-Trichinella fusion gene, Te1" / Chapter 3.1 --- Rationale of the gene construction --- p.51 / Chapter 3.2 --- Isolation of T. spiralis P49 gene by cDNA amplification --- p.55 / Chapter 3.3 --- Cloning of Trichinella P49 cDNA to g4R --- p.58 / Chapter 3.4 --- Screening of recombinant clones --- p.58 / Chapter VII. --- Chapter4 Characterization of Tc1 fusion gene product / Chapter 4.1 --- Transfection of fusion gene to J558L myeloma cells / Chapter 4.2 --- Antigenicity of fusion gene product with respect to Trichinella activity --- p.64 / Chapter 4.3 --- Detection of Ig CH2-CH3 domains in fusion gene product --- p.65 / Chapter 4.4 --- Size determination of fusion gene product --- p.69 / Chapter 4.5 --- Transcriptional and genomic analysis of transfectants producing the fusion gene product --- p.71 / Chapter 4.5.1 --- Genomic analysis --- p.71 / Chapter 4.5.2 --- Sequence analysis of transcript from the CH1 to CH2 exon --- p.71 / Chapter 4.5.3 --- Sequence analysis of transcript from the CH1 to CH3 exon --- p.77 / Chapter VIII. --- "Chapter5 Construction and characterization of second fusion gene, Tc2, using modified g4R vector" --- p.81 / Chapter IX. --- Chapter6 General Discussion / Use of recombinant DNA technology to produceantigen for use in the diagnosis of infectious diseases --- p.89 / Characterization of the fusion gene product --- p.89 / Absence of Trichinella sequence in fusion gene product due to exon skipping --- p.94 / A new strategy for producing Ig fusion proteins: modification of the g4R vector --- p.96 / Prospect of utilizing Ig expression system for producing antigen --- p.97 / Chapter X. --- References --- p.99 / Chapter XI. --- Appendix --- p.107
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318152 |
| Date | January 1994 |
| Contributors | Fu, Kit Yu., Chinese University of Hong Kong Graduate School. Division of Medical Sciences. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiii, 107 leaves : ill. (some mounted) ; 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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