Plant as bioreactor: transgenic expression of malaria surface antigen in plants.

by Ng Wang Kit.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 131-139).
s in English and Chinese.
Acknowledgements --- p.iii
p.v
List of Tables --- p.ix
List of Figures --- p.x
List of Abbreviations --- p.xiii
Table of Contents --- p.xv
Chapter Chapter 1: --- General Introduction --- p.1
Chapter Chapter 2: --- Literature Review --- p.3
Chapter 2.1 --- Malaria --- p.3
Chapter 2.1.1 --- Global picture --- p.3
Chapter 2.1.2 --- Malaria mechanics --- p.4
Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4
Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5
Chapter 2.2.1 --- Antimalarial drugs --- p.5
Chapter 2.2.2 --- Drug resistance --- p.6
Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7
Chapter 2.3.1 --- Malarial vaccine developments --- p.7
Chapter 2.3.2 --- Transmission blocking vaccines --- p.7
Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9
Chapter 2.3.4 --- Blood stage vaccines --- p.10
Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11
Chapter 2.5 --- Plants as bioreactors --- p.12
Chapter 2.5.1 --- Products of transgenic plants --- p.13
Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14
Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15
Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15
Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16
Chapter 2.7.1 --- Arabidopsis --- p.16
Chapter 2.7.2 --- Tobacco --- p.17
Chapter 2.8 --- Transformation methods --- p.17
Chapter 2.8.1 --- Direct DNA uptake --- p.17
Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17
Chapter 2.8.1.2 --- Biolistic transformation --- p.18
Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18
Chapter 2.8.2.1 --- Leaf-disc technique --- p.18
Chapter 2.8.2.2 --- In planta transformation --- p.19
Chapter 2.9 --- Phaseolin --- p.20
Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21
Chapter 2.11 --- Aims of study and hypotheses --- p.22
Chapter Chapter 3: --- Materials and Methods --- p.24
Chapter 3.1 --- Introduction --- p.24
Chapter 3.2 --- Chemicals --- p.24
Chapter 3.3 --- Expression in tobacco system --- p.24
Chapter 3.3.1 --- Plant materials --- p.24
Chapter 3.3.2 --- Bacterial strains --- p.25
Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25
Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26
Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30
Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30
Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33
Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34
Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35
Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36
Chapter 3.3.8 --- GUS assay --- p.37
Chapter 3.3.9 --- Genomic DNA isolation --- p.37
Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38
Chapter 3.3.11 --- Southern blot analysis --- p.38
Chapter 3.3.12 --- Total seeds RNA isolation --- p.39
Chapter 3.3.13 --- RT-PCR --- p.39
Chapter 3.3.14 --- Northern blot analysis --- p.40
Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40
Chapter 3.3.16 --- Western blot analysis --- p.41
Chapter 3.4 --- Expression in Arabidopsis system --- p.42
Chapter 3.4.1 --- Plant materials --- p.42
Chapter 3.4.2 --- Bacterial strains --- p.42
Chapter 3.4.3 --- Chimeric gene construction --- p.42
Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43
Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45
Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45
Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45
Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49
Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49
Chapter 3.4.7 --- Arabidopsis Transformation --- p.49
Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50
Chapter 3.4.9 --- Selection of successful transformants --- p.51
Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51
Chapter 3.4.11 --- GUS assay --- p.52
Chapter 3.4.12 --- Genomic DNA isolation --- p.52
Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52
Chapter 3.4.14 --- Southern Blot analysis --- p.52
Chapter 3.4.15 --- Total siliques RNA isolation --- p.53
Chapter 3.4.16 --- RT-PCR --- p.53
Chapter 3.4.17 --- Northern blot analysis --- p.53
Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54
Chapter 3.4.18 --- Western blot analysis --- p.54
Chapter 3.5 --- In vitro transcription and translation --- p.54
Chapter 3.5.1 --- In vitro transcription --- p.54
Chapter 3.5.2 --- In vitro translation --- p.55
Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56
Chapter 3.6.1 --- Chimeric gene constructs --- p.56
Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61
Chapter Chapter 4: --- Results --- p.63
Chapter 4.1 --- Tobacco system --- p.63
Chapter 4.1.1 --- Chimeric gene constructs --- p.63
Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65
Chapter 4.1.3 --- GUS activity assay --- p.67
Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68
Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68
Chapter 4.1.4.2 --- Southern blot analysis --- p.70
Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72
Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72
Chapter 4.1.5.2 --- Northern blot analysis --- p.75
Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76
Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78
Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78
Chapter 4.1.7.2 --- Western blot analysis --- p.78
Chapter 4.2 --- Arabidopsis system --- p.83
Chapter 4.2.1 --- Chimeric gene constructs --- p.83
Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85
Chapter 4.2.3 --- Selection of transgenic plants --- p.87
Chapter 4.2.4 --- Assay of GUS activity --- p.91
Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92
Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92
Chapter 4.2.5.2 --- Southern blot analysis --- p.96
Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99
Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99
Chapter 4.2.6.2 --- Northern blot analysis --- p.106
Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107
Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108
Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108
Chapter 4.2.8.2 --- Western blot analysis --- p.108
Chapter 4.3 --- In vitro transcription and translation --- p.112
Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115
Chapter Chapter 5: --- Discussion and Future perspectives --- p.117
Chapter 5.1 --- Failure in detecting transgene expression --- p.117
Chapter 5.2 --- Poor transgene expression --- p.120
Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120
Chapter 5.2.2 --- AT-richness --- p.121
Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123
Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125
Chapter 5.2.5 --- Codon usage --- p.126
Chapter 5.3 --- Future perspectives --- p.127
Chapter Chapter 6: --- Conclusion --- p.129
References --- p.131
http://library.cuhk.edu.hk/record=b5895896
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