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Production of Trichinella spiralis antigen as a recombinant fusion protein of immunoglobulin.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318152
Date January 1994
ContributorsFu, Kit Yu., Chinese University of Hong Kong Graduate School. Division of Medical Sciences.
PublisherChinese University of Hong Kong
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xiii, 107 leaves : ill. (some mounted) ; 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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