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Expression and characterization of a human lysosomal enzyme α-iduronidase in tobacco BY-2 cells. / Expression & characterization of a human lysosomal enzyme α-iduronidase in tobacco BY-2 cells / Expression and characterization of a human lysosomal enzyme alpha-iduronidase in tobacco BY-2 cells

Fu Lai Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 106-110). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Lists of Figures --- p.x / Lists of Tables --- p.xiii / List of Abbreviations --- p.xiv / Amino acid abbreviation --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Human α-L-iduronidase (hIDUA) --- p.2 / Chapter 1.1.1 --- Lysosomal storage disease --- p.2 / Chapter 1.1.2 --- Treatments of MPS 1 --- p.4 / Chapter 1.2 --- Plant cells as bioreactors --- p.5 / Chapter 1.3 --- The Plant secretary pathway --- p.7 / Chapter 1.3.1 --- Transport of soluble proteins --- p.9 / Chapter 1.3.2 --- Transport of integral membrane proteins --- p.10 / Chapter 1.4 --- Differences between plant and human proteins --- p.11 / Chapter 1.5 --- Reducing the differences between plant and human proteins --- p.12 / Chapter 1.6 --- Previous study: Expression of IDUA in transgenic tobacco plant --- p.13 / Chapter 1.7 --- Project objectives --- p.14 / Chapter 1.8 --- Long term significance --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials --- p.18 / Chapter 2.2.1 --- Chemical --- p.18 / Chapter 2.2.2 --- Plant materials --- p.18 / Chapter 2.2.3 --- Plasmid vectors and bacterial strains --- p.18 / Chapter 2.2.4 --- Human a-iduronidase (hIDUA) cDNA --- p.19 / Chapter 2.2.5 --- Primers --- p.20 / Chapter 2.3 --- Methods --- p.22 / Chapter 2.3.1 --- Generation of IDUA antibodies --- p.22 / Chapter 2.3.1.1 --- Synthetic peptide raised IDUA antibodies --- p.23 / Chapter 2.3.1.1.1 --- Design of synthetic peptides --- p.23 / Chapter 2.3.1.1.2 --- Immunization of rabbits --- p.25 / Chapter 2.3.1.2 --- E. coli-derived rhIDUA protein --- p.25 / Chapter 2.3.1.2.1 --- Cloning and expression of rhIDUA --- p.25 / Chapter 2.3.1.2.2 --- Western analysis of E. coli-derived rhIDUA --- p.29 / Chapter 2.3.1.2.3 --- MS/MS analysis of rhIDUA protein --- p.29 / Chapter 2.3.1.2.4 --- Immunization of rabbits --- p.31 / Chapter 2.3.2 --- Affinity-purified antibodies --- p.33 / Chapter 2.3.3 --- Characterization of affinity-purified IDUA antibodies --- p.33 / Chapter 2.3.4 --- Construction of chimeric gene constructs --- p.34 / Chapter 2.3.5 --- Expression of IDUA in tobacco BY-2 cells --- p.39 / Chapter 2.3.5.1 --- Electropoartion of Agrobacteria --- p.39 / Chapter 2.3.5.2 --- Agrobacterium-mediated transformation --- p.39 / Chapter 2.3.5.3 --- Screening of positive trans formants --- p.40 / Chapter 2.3.6 --- Characterization of transgenic BY-2 cell expressing IDUA fusion --- p.40 / Chapter 2.3.6.1 --- Genomic DNA polymerase chain reaction (Genomic DNA PCR) --- p.40 / Chapter 2.3.6.1.1 --- Genomic DNA extraction from BY-2 callus --- p.40 / Chapter 2.3.6.1.2 --- Genomic DNA PCR of tobacco BY-2 callus --- p.41 / Chapter 2.3.6.2 --- Reverse transcription-PCR (RT-PCR) --- p.42 / Chapter 2.3.6.2.1 --- Total RNA extraction from BY-2 cell --- p.42 / Chapter 2.3.6.2.2 --- RT-PCR of BY-2 cell --- p.42 / Chapter 2.3.6.3 --- Western blot analysis of BY-2 cell and medium --- p.43 / Chapter 2.3.6.3.1 --- Protein extraction from tobacco BY-2 cells and culture medium --- p.43 / Chapter 2.3.6.3.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 2.3.6.3.3 --- Immunodetection and Coomassie blue stain --- p.44 / Chapter 2.3.7 --- Purification of IDUA from culture media --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Generation of IDUA antibodies --- p.48 / Chapter 3.1.1 --- Cloning and expression of rhIDUA in E. coli --- p.48 / Chapter 3.1.2 --- Characterization of IDUA antibodies --- p.51 / Chapter 3.1.2.1 --- Specificity of IDUA antibodies towards hIDUA protein. --- p.51 / Chapter 3.1.2.2 --- Cross-reactivity of IDUA antibodies with wild type tobacco BY-2 cell --- p.55 / Chapter 3.2 --- Chimeric gene constructs construction and confirmation --- p.58 / Chapter 3.3 --- Screening of transformed tobacco BY-2 callus with kanamycin-resistance --- p.66 / Chapter 3.4 --- Genomic DNA PCR screening of transformed tobacco BY-2 callus . --- p.67 / Chapter 3.5 --- RT-PCR screening of transformed BY-2 cells --- p.70 / Chapter 3.6 --- Western blot analysis of transformed tobacco BY-2 cells and culture media --- p.72 / Chapter 3.6.1 --- Tobacco BY-2 cells --- p.72 / Chapter 3.6.2 --- Tobacco BY-2 cell culture media --- p.76 / Chapter 3.7 --- Purification of IDUA protein in culture media --- p.81 / Chapter Chapter 4 --- Discussion --- p.82 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.89 / Chapter 5.1 --- Summary --- p.90 / Chapter 5.2 --- Future perspectives --- p.92 / Appendix Identification and Characterization of an Unknown Protein by 1B Antibody --- p.93 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Objectives --- p.94 / Chapter 6.3 --- Materials and Methods --- p.95 / Chapter 6.3.1 --- Western blot analysis of different plant species --- p.95 / Chapter 6.3.2 --- Subcellular localization of the unknown protein --- p.95 / Chapter 6.3.3 --- Affinity-purification of the unknown protein --- p.95 / Chapter 6.4 --- Results --- p.97 / Chapter 6.4.1 --- Western blot analysis of different plant species --- p.97 / Chapter 6.4.2 --- Subcellular localization of an unknown protein --- p.98 / Chapter 6.4.3 --- Affinity-purification of 1B protein --- p.104 / Chapter 6.5 --- Summary and Future Perspectives --- p.105 / Chapter 6.5.1 --- Summary --- p.105 / Chapter 6.5.2 --- Future Perspectives --- p.105 / References --- p.106

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325737
Date January 2006
ContributorsFu, Lai Hong., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvi, 110 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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