Return to search

Membrane anchor for vacuolar targeting: expression of a human lysosomal enzyme iduronidase (hIDUA) in transgenic tobacco plants.

Seto Tai Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 122-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.v / Abstract (in Chinese) --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xvi / List of Figures --- p.xv / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Tobacco seed as bioreactor --- p.4 / Chapter 1.2.1 --- Advantages of using tobacco seed to produce bioactive human lysosomal enzyme --- p.4 / Chapter 1.2.2 --- Disadvantages and potential problems of using tobacco seed to produce bioactive human lysosomal enzyme --- p.5 / Chapter 1.2.2.1 --- Difference of asparagine-linked N-glycosylation between plant and human protein --- p.8 / Chapter 1.2.2.2 --- Immunogenicity of recombinant protein with plant-derived N-glycan to human --- p.10 / Chapter 1.2.2.3 --- "Strategy to ""humanize"" plant-derived recombinant human lysosomal enzyme" --- p.10 / Chapter 1.2.2.4 --- Lack of specific glycan structure一mannose-6-phosphate (M6P) tag addition --- p.11 / Chapter 1.2.2.5 --- Strategy for M6P tag addition on plant-derived human lysosomal enzyme --- p.12 / Chapter 1.3 --- The plant secretory pathway --- p.13 / Chapter 1.3.1 --- Plant vacuole in tobacco seed --- p.16 / Chapter 1.3.2 --- Soluble protein trafficking in plant cell --- p.17 / Chapter 1.3.3 --- Integral membrane protein trafficking in plant cell --- p.17 / Chapter 1.3.4 --- Components involved in integral membrane protein trafficking to PSV crystalloid --- p.19 / Chapter 1.3.4.1 --- BP-80 (80-kDa binding protein) --- p.19 / Chapter 1.3.4.2 --- α-TIP (α-tonoplast intrinsic protein) --- p.20 / Chapter 1.3.5 --- Using specific integral membrane protein trafficking system to target recombinant human lysosomal enzyme to tobacco seed PSV --- p.21 / Chapter 1.4 --- Homo sapiens α-L-iduronidase (hIDUA) --- p.21 / Chapter 1.4.1 --- Global situation of lysosomal storage disease一hIDUA deficiency --- p.21 / Chapter 1.4.2 --- Physiological role --- p.22 / Chapter 1.4.3 --- Molecular property --- p.24 / Chapter 1.4.3.1 --- Mutation and polymorphism --- p.24 / Chapter 1.4.4 --- Lysosomal secretory pathway --- p.24 / Chapter 1.4.5 --- Biochemical property --- p.25 / Chapter 1.4.6 --- Clinical application --- p.27 / Chapter 1.4.6.1 --- Enzyme replacement therapy (ERT) --- p.27 / Chapter 1.4.6.2 --- Clinical trial --- p.28 / Chapter 1.4.6.3 --- Economic value --- p.29 / Chapter 1.4.7 --- Expression system --- p.29 / Chapter 1.4.7.1 --- Production (overexpression) of rhIDUA in CHO cell system --- p.30 / Chapter 1.4.7.2 --- Production of rhIDUA in tobacco plant leaf --- p.30 / Chapter 1.5 --- Project objective and long-term significance --- p.30 / Chapter 1.5.1 --- Project objective --- p.30 / Chapter 1.5.2 --- Long-term significance --- p.31 / Chapter Chapter 2 --- Generation and Characterization of Anti-IDUA Antibodies --- p.32 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Chemical --- p.33 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Generation of polyclonal anti-IDUA antibody --- p.35 / Chapter 2.3.1.1 --- Design of synthetic peptide --- p.35 / Chapter 2.3.1.2 --- Conjugation of synthetic peptide to carrier protein --- p.39 / Chapter 2.3.1.3 --- Immunization of rabbit --- p.39 / Chapter 2.3.2 --- Characterization of polyclonal anti-IDUA antibody in rabbit serum --- p.40 / Chapter 2.3.2.1 --- Dot-blot analysis --- p.40 / Chapter 2.3.3 --- Purification of polyclonal anti-IDUA antibody --- p.42 / Chapter 2.3.3.1 --- Construction of anti-IDUA antibody affinity column --- p.42 / Chapter 2.3.3.2 --- Affinity-purification of anti-IDUA antibody --- p.42 / Chapter 2.3.4 --- Western blot detection of denatured rhIDUA --- p.42 / Chapter 2.4 --- Results --- p.43 / Chapter 2.4.1 --- Characterization of polyclonal anti-IDUA antibody --- p.43 / Chapter 2.5 --- Discussion --- p.51 / Chapter 2.6 --- Conclusion --- p.51 / Chapter Chapter 3 --- Generation and Characterization of Transgenic Tobacco Plants Expressing rhIDUA Fusions --- p.52 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Signal peptide of hIDUA (hIDUA SP) --- p.54 / Chapter 3.1.2 --- Signal peptide of proaleurain (Pro. SP) --- p.54 / Chapter 3.1.3 --- Hypothesis to be tested in this study --- p.54 / Chapter 3.2 --- Materials --- p.55 / Chapter 3.2.1 --- Chemical --- p.55 / Chapter 3.2.2 --- Primers --- p.55 / Chapter 3.2.3 --- Bacterial strain --- p.58 / Chapter 3.2.4 --- The insert-Homo sapiens α-L-iduronidase (hIDUA) cDNA used in this study --- p.58 / Chapter 3.2.5 --- The vector-pLJ526 used in this study --- p.59 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Construction of chimeric gene construct --- p.61 / Chapter 3.3.1.1 --- Restriction endonuclease´ؤPfIMIl --- p.61 / Chapter 3.3.1.2 --- Recombinant DNA and molecular cloning techniques used in this study --- p.61 / Chapter 3.3.1.3 --- Cloning of pSPIDUA-FLAG --- p.62 / Chapter 3.3.1.4 --- Cloning of pSPIDUA-control --- p.62 / Chapter 3.3.1.5 --- Cloning of a universal construct (pUniversal) --- p.62 / Chapter 3.3.1.6 --- Cloning of pSP-IDUA-T7 --- p.66 / Chapter 3.3.1.7 --- Cloning of pSP-IDUA-control --- p.66 / Chapter 3.3.1.8 --- Cloning of chimeric gene construct into Agrobacterium binary vector --- p.66 / Chapter 3.3.2 --- Expression of chimeric gene construct in tobacco plant --- p.73 / Chapter 3.3.2.1 --- Tobacco plant --- p.73 / Chapter 3.3.2.2 --- Electroporation of Agrobacterium --- p.73 / Chapter 3.3.2.3 --- Agrobacterium-mediated transformation of tobacco plant --- p.74 / Chapter 3.3.2.4 --- Selection and regeneration of tobacco transformant --- p.75 / Chapter 3.3.3 --- Characterization of transgenic tobacco plant expressing rhIDUA fusion --- p.75 / Chapter 3.3.3.1 --- Genomic DNA polymerase chain reaction (PCR) --- p.75 / Chapter 3.3.3.2 --- Southern blot analysis --- p.76 / Chapter 3.3.3.3 --- Total RNA reverse transcription-PCR (RT-PCR) --- p.77 / Chapter 3.3.3.4 --- Northern blot analysis of tobacco leaf --- p.78 / Chapter 3.3.3.5 --- Western blot analysis --- p.79 / Chapter 3.3.4 --- Purification of plant-derived rhIDUA fusion --- p.81 / Chapter 3.3.4.1 --- Construction of affinity column with anti-IDUA antibody --- p.81 / Chapter 3.3.4.2 --- Affinity-purification of rhIDUA fusion from tobacco mature seed --- p.81 / Chapter 3.3.5 --- Confocal immunoflorescence study --- p.82 / Chapter 3.3.5.1 --- Preparation of paraffin section --- p.82 / Chapter 3.3.5.2 --- Single immunocytochemical labeling --- p.82 / Chapter 3.3.5.3 --- Double labeling with one monoclonal and one polyclonal antibodies --- p.83 / Chapter 3.3.5.4 --- Double labeling with two polyclonal antibodies --- p.83 / Chapter 3.3.5.5 --- Image collection --- p.84 / Chapter 3.4 --- Results --- p.85 / Chapter 3.4.1 --- Chimeric gene construction and confirmation --- p.85 / Chapter 3.4.2 --- Selection and regeneration of tobacco transformant with kanamycin- resistance --- p.86 / Chapter 3.4.3 --- Genomic DNA PCR screening of tobacco transformant --- p.88 / Chapter 3.4.4 --- Southern blot analysis of tobacco transformant --- p.91 / Chapter 3.4.5 --- Total RNA RT-PCR screening of tobacco transformant --- p.93 / Chapter 3.4.6 --- Northern blot analysis of tobacco transformant --- p.93 / Chapter 3.4.7 --- Western blot analysis --- p.96 / Chapter 3.4.7.1 --- Western blot analysis of pSP-IDUA-T7-121 transformant leaf --- p.96 / Chapter 3.4.7.2 --- Western blot analysis of pSP-IDUA-T7-121 transformant mature seed --- p.98 / Chapter 3.4.8 --- Affinity-purification of rhIDUA fusion --- p.98 / Chapter 3.4.9 --- Expression level of rhIDUA fusion --- p.102 / Chapter 3.4.10 --- Subcellular localization of rhIDUA fusion --- p.102 / Chapter 3.5 --- Discussion --- p.111 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.117 / References --- p.122 / Appendix 1 --- p.139 / Appendix II (List of Abbreviations) --- p.141

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325156
Date January 2005
ContributorsSeto, Tai Chi., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvii, 145 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/)

Page generated in 0.0031 seconds