Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV):
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| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
2000
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Aachen, Techn. Hochsch., Diss., 2000 |
| Physical Description: | 125 S. graph. Darst. : 21 cm |
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MARC
| LEADER | 00000nam a2200000 c 4500 | ||
|---|---|---|---|
| 001 | BV014851617 | ||
| 003 | DE-604 | ||
| 005 | 20030403 | ||
| 007 | t | ||
| 008 | 021024s2000 gw d||| m||| 00||| eng d | ||
| 016 | 7 | |a 965200868 |2 DE-101 | |
| 035 | |a (OCoLC)633954946 | ||
| 035 | |a (DE-599)BVBBV014851617 | ||
| 040 | |a DE-604 |b ger |e rakddb | ||
| 041 | 0 | |a eng | |
| 044 | |a gw |c DE | ||
| 049 | |a DE-355 |a DE-83 | ||
| 100 | 1 | |a Zhang, Mei-Yun |d 1965- |e Verfasser |0 (DE-588)124054706 |4 aut | |
| 245 | 1 | 0 | |a Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) |c vorgelegt von Mei-Yun Zhang |
| 264 | 1 | |c 2000 | |
| 300 | |a 125 S. |b graph. Darst. : 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Aachen, Techn. Hochsch., Diss., 2000 | ||
| 650 | 0 | 7 | |a Tomatenbronzefleckenvirus |0 (DE-588)4308006-6 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Leichte Kette |0 (DE-588)4502527-7 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Rekombinantes Protein |0 (DE-588)4277353-2 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Antikörper |0 (DE-588)4002290-0 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Tomatenbronzefleckenvirus |0 (DE-588)4308006-6 |D s |
| 689 | 0 | 1 | |a Antikörper |0 (DE-588)4002290-0 |D s |
| 689 | 0 | 2 | |a Rekombinantes Protein |0 (DE-588)4277353-2 |D s |
| 689 | 0 | 3 | |a Leichte Kette |0 (DE-588)4502527-7 |D s |
| 689 | 0 | |5 DE-604 | |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-010046762 | |
Record in the Search Index
| _version_ | 1807683162870906880 |
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| adam_text |
CONTENTS
PAGE
1.
INTRODUCTION
1
1.1
TSWV
GENOME
AND
ITS
INTERACTION
WITH
PLANTS
2
1.2
PATHOGEN-DERIVED
RESISTANCE
TO
TSWV
4
1.3
DISADVANTAGES
OF
PATHOGEN-DERIVED
RESISTANCE
5
1.4
PLANTIBODY-MEDIATED
RESISTANCE
6
1.5
ANTIBODY
PHAGE
DISPLAY
TECHNOLOGY
8
1.5.1
ANTIBODY
LIBRARIES
9
1.5.2
ANTIBODY
DISPLAY
METHODOLOGY
10
1.5.3
PHAGE-DISPLAYED
ANTIBODY
SELECTION
12
1.5.4
PHAGE
ANTIBODY
SCREENING
14
1.6
CHICKEN
ANTIBODY
REPERTOIRE
14
1.7
ABOUT
THIS
THESIS
15
2.
MATERIALS
18
2.1
CHEMICALS
AND
CONSUMABLES
18
2.2
BUFFERS,
MEDIA
AND
SOLUTIONS
18
2.3
MATRIX
AND
MEMBRANES
18
2.4
ENZYMES
AND
KITS
19
2.5
PRIMARY
ANTIBODIES,
SECONDARY
ANTIBODIES
AND
SUBSTRATES
19
2.6
VECTORS
20
2.7
BIOLOGICAL
MATERIALS
21
2.8
OLIGONUCLEOTIDES
22
2.9
EQUIPMENT
25
3.
METHODS
27
3.1
RECOMBINANT
DNA
TECHNOLOGIES
27
3.1.1
DNA
EXTRACTION
FROM
AGAROSE
GEL
27
3.1.1.1
DNA
ELUTION
FROM
DEAE
PAPER
27
3.1.1.2
DNA
RECOVERY
FROM
AGAROSE
GEL
28
3.1.2
TRANSFORMATION
OF
BACTERIA
28
3.1.2.1
PREPARATION
OF
E.
COLI
COMPETENT
CELLS
FOR
HEAT-SHOCK
TRANSFORMATION
28
3.1.2.2
TRANSFORMATION
OF
E.
COLI
BY
HEAT-SHOCK
29
3.1.2.3
PREPARATION
OF
E.
COLI
COMPETENT
CELLS
FOR
ELECTROPORATION
29
3.1.2.4
TRANSFORMATION
OF
E.
COLI
BY
ELECTROPORATION
30
3.1.2.5
PREPARATION
OF
AGROBACTERIUM
COMPETENT
CELLS
FOR
ELECTROPORATION
30
3.1.2.6
TRANSFORMATION
OF
AGROBACTERIUM
BY
ELECTROPORATION
30
3.1.2.7
PREPARATION
OF
AGROBACTERIUM
COMPETENT
CELLS
FOR
N2
TRANSFORMATION
31
3.1.2.8
N2
TRANSFORMATION
OF
AGROBACTERIUM
31
3.1.3
PREPARATION
OF
PLASMID
DNA
32
3.1.3.1TELT
METHOD
32
3.1.3.2STELT
METHOD
32
3.1.3.3
PLASMID
DNA
PREPARATION
KIT
33
3.1.4
PCR
AMPLIFICATION
33
3.1.5
DNA
SEQUENCING
34
3.2
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
PROTEINS
34
3.2.1
EXPRESSION
AND
PURIFICATION
OF
GST
FUSION
PROTEINS
34
3.2.2
EXPRESSION
AND
PURIFICATION
OF
CBD
FUSION
PROTEINS
35
3.2.3
EXPRESSION
AND
PURIFICATION
OF
SOLUBLE
SCFV
ANTIBODIES
36
3.2.3.1
MINI-SCALE
EXPRESSION
OF
SCFVS
IN
PHEN4II
PHAGEMID
VECTOR
36
3.2.3.2
LARGE-SCALE
EXPRESSION
OF
SCFVS
IN
PHEN4H
PHAGEMID
VECTOR
AND
PREPARATION
OF
PERIPLASMIC
EXTRACTS
BY
OSMOTIC
SHOCK
36
3.2.3.3
LARGE-SCALE
EXPRESSION
OF
SCFVS
IN
PTMZL
VECTOR
AND
PURIFICATION
OF
SECRETED
SCFVS
BY
IMAC
37
3.3
PROTEIN
ANALYSIS
38
3.3.1
QUANTIFICATION
OF
TOTAL
SOLUBLE
PROTEINS
38
3.3.2
SDS-PAGE
38
3.3.3
IMMUNOBLOT
39
3.3.4
DOT
BLOT
ANALYSIS
39
3.3.5
ELISA
(ENZYME-LINKED
IMMUNOSORBENT
ASSAY)
39
3.4 IMMUNIZATION
OF
ANIMALS
41
3.4.1
IMMUNIZATION
OF
MICE
41
3.4.2
IMMUNIZATION
OF
CHICKEN
41
3.4.3
ISOLATION
OF
CHICKEN
POLYCLONAL
ANTIBODIES
FROM
EGG
YOLK
41
3.5 CONSTRUCTION
OF
PHAGE-DISPLAYED
SCFV
LIBRARIES
41
3.5.1
ISOLATION
OF
TOTAL
RNA
AND
MRNA
FROM
SPLEEN
CELLS
41
3.5.2
CONSTRUCTION
OF
SCFV
LIBRARIES
41
3.6
PHAGE
LIBRARY
PANNING
42
3.6.1
BIOTINYLATION
OF
ANTIGENS
42
3.6.2
DETERMINATION
OF
BIOTINYLATION
EFFICIENCY
BY
DOT-BLOT
42
3.6.3
SOLUTION-PHASE
PANNING
WITH
BIOTINYLATED
ANTIGENS
43
3.6.4
SOLID-PHASE
PANNING
ON
PVDF
MEMBRANE
43
3.7
CHARACTERIZATION
OF
PHAGE-DISPLAYED
SCFVS
(PHAGE
ELISA)
44
3.7.1
INDIRECT
PHAGE
ELISA
44
3.7.2
CAPTURE
PHAGE
ELISA
44
3.7.3
CROSS-REACTIVITY
ANALYSIS
45
3.8
GENERATION
AND
CHARACTERIZATION
OF
TRANSGENIC
PLANTS
45
3.8.1
TRANSIENT
EXPRESSION
OF
SCFVS
IN
TOBACCO
LEAVES
BY
VACUUM
INFILTRATION
45
3.8.1.1
PREPARATION
OF
AGROBACTERIUM
45
3.8.1.2
INFILTRATION
OF
INTACT
LEAVES
45
3.8.2
AGROBACTERIUM-MEDIATED
STABLE
TRANSFORMATION
OF
TOBACCO
PLANTS
46
3.8.3
PREPARATION
OF
TOTAL
SOLUBLE
PROTEINS
FROM
PLANT
LEAVES
47
4.
RESULTS
49
4.1
CODON
OPTIMIZATION
OF
THREE
NS
M
DOMAINS
49
4.2
EXPRESSION
AND
PURIFICATION
OF
RECOMBINANT
PROTEINS
52
4.2.1
GST
FUSION
PROTEINS
52
4.2.2
CBD
FUSION
PROTEINS
54
4.2.3
FULL-LENGTH
NS
M
PROTEIN
54
4.3
IMMUNIZATION
OF
ANIMALS
AND
DETERMINATION
OF
ANTIBODY
TITER
58
4.4
CONSTRUCTION
OF
PHAGEMID-SCFV
LIBRARIES
59
4.4.1
CONSTRUCTION
OF
PHAGEMID
VECTOR
PHEN4IN
59
4.4.2
ISOLATION
OF
TOTAL
RNA
AND
MRNA
FROM
SPLEEN
CELLS
61
4.4.3
CONSTRUCTION
OF
PHAGEMID-SCFV
LIBRARIES
61
4.5
SELECTION
AND
CHARACTERIZATION
OF
SPECIFIC
PHAGE
CLONES
63
4.5.1
ISOLATION
OF
DOMAIN-SPECIFIC
PHAGE
CLONES
USING
GST
FUSION
PROTEINS
63
4.5.1.1
SOLUTION-PHASE
PANNING
AGAINST
GST
FUSION
PROTEINS
63
4.5.1.2
CHARACTERIZATION
OF
MONOCLONAL
PHAGE
BY
PHAGE
ELISA
64
4.5.1.3
SEQUENCE
ANALYSIS
OF
UNSELECTED
AND
SELECTED
PHAGE
CLONES
65
4.5.1.4
MINI-SCALE
EXPRESSION
AND
CHARACTERIZATION
OF
ISOLATED
CLONES
USING
SOLUBLE
SCFVS
67
4.5.2
ISOLATION
OF
NSM-SPECIFIC
PHAGE
CLONES
USING
FULL-LENGTH
NS
M
PROTEIN
70
4.6
CONSTRUCTION
OF
PROKARYOTIC
EXPRESSION
PTMZL
VECTOR
71
74
4.7
EXPRESSION
AND
CHARACTERIZATION
OF
SOLUBLE
SCFVS
4.7.1
LARGE-SCALE
EXPRESSION
OF
SOLUBLE
SCFVS
74
4.7.2 IMMUNOBLOT
ANALYSIS
OF
PURIFIED
SOLUBLE
SCFVS
75
4.7.3
ELISA
ANALYSIS
OF
PURIFIED
SOLUBLE
SCFVS
76
4.8 GENERATION
AND
CHARACTERIZATION
OF
TRANSGENIC
PLANTS
78
4.8.1
CLONING
OF
NSM-SPECIFIC
SCFV
GENES
INTO
PLANT
EXPRESSION
PSSHL
VECTOR
78
4.8.2
TRANSIENT
EXPRESSION
OF
SCFVS
IN
TOBACCO
LEAVES
BY
VACUUM
INFILTRATION
80
4.8.3
STABLE
TRANSFORMATION
AND
ANALYSIS
OF
TRANSGENIC
PLANTS
83
5.
DISCUSSION
86
S.L
EXPRESSION
OF
"
RARE
"
CODON-RICH
NS
M
GENE
IN
E.
COLI
86
5.2
OPTIMIZATION
OF
CODON
USAGE
AND
EXPRESSION
OF
THREE
NS
M
DOMAINS
88
5.3
GENERATION
OF
SPECIFIC
SCFVS
BY
PHAGE
DISPLAY
89
5.3.1
PHAGE-DISPLAYED
ANTIBODY
SELECTION
USING
GST
FUSION
PROTEIN
89
5.3.2
PHAGE-DISPLAYED
ANTIBODY
SELECTION
USING
A
PVDF
MEMBRANE
AS
A
SUPPORT
92
5.3.3
CHARACTERIZATION
OF
ISOLATED
CLONES
USING
SOLUBLE
SCFVS
94
5.3.4
CHICKEN-DERIVED
SCFVS
95
5.4
PLANTIBODY-MEDIATED
VIRUS
RESISTANCE
95
5.4.1
TARGETS
FOR
PLANTIBODIES
95
5.4.2
ANTIBODY
EXPRESSION
IN
PLANT
CYTOSOL
97
5.4.3
TRANSIENT
GENE
EXPRESSION
AND
STABLE
TRANSFORMATION
99
5.5
FUTURE
PROSPECTS
100
6.
SUMMARY
103
7.
REFERENCES
105
8.
APPENDICES
122
8.1
ABBREVIATIONS
122
8.2
FIGURES
124
8.3
TABLES
125 |
| any_adam_object | 1 |
| author | Zhang, Mei-Yun 1965- |
| author_GND | (DE-588)124054706 |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV014851617 |
| illustrated | Illustrated |
| indexdate | 2024-08-18T00:30:04Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-010046762 |
| oclc_num | 633954946 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-83 |
| owner_facet | DE-355 DE-BY-UBR DE-83 |
| physical | 125 S. graph. Darst. : 21 cm |
| publishDate | 2000 |
| publishDateSearch | 2000 |
| publishDateSort | 2000 |
| record_format | marc |
| spelling | Zhang, Mei-Yun 1965- Verfasser (DE-588)124054706 aut Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) vorgelegt von Mei-Yun Zhang 2000 125 S. graph. Darst. : 21 cm txt rdacontent n rdamedia nc rdacarrier Aachen, Techn. Hochsch., Diss., 2000 Tomatenbronzefleckenvirus (DE-588)4308006-6 gnd rswk-swf Leichte Kette (DE-588)4502527-7 gnd rswk-swf Rekombinantes Protein (DE-588)4277353-2 gnd rswk-swf Antikörper (DE-588)4002290-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Tomatenbronzefleckenvirus (DE-588)4308006-6 s Antikörper (DE-588)4002290-0 s Rekombinantes Protein (DE-588)4277353-2 s Leichte Kette (DE-588)4502527-7 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010046762&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Zhang, Mei-Yun 1965- Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) Tomatenbronzefleckenvirus (DE-588)4308006-6 gnd Leichte Kette (DE-588)4502527-7 gnd Rekombinantes Protein (DE-588)4277353-2 gnd Antikörper (DE-588)4002290-0 gnd |
| subject_GND | (DE-588)4308006-6 (DE-588)4502527-7 (DE-588)4277353-2 (DE-588)4002290-0 (DE-588)4113937-9 |
| title | Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) |
| title_auth | Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) |
| title_exact_search | Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) |
| title_full | Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) vorgelegt von Mei-Yun Zhang |
| title_fullStr | Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) vorgelegt von Mei-Yun Zhang |
| title_full_unstemmed | Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) vorgelegt von Mei-Yun Zhang |
| title_short | Generation, characterization and expression of single chain antibody fragments (scFvs) specific for the NSM movement protein of tomato spotted virus (TSWV) |
| title_sort | generation characterization and expression of single chain antibody fragments scfvs specific for the nsm movement protein of tomato spotted virus tswv |
| topic | Tomatenbronzefleckenvirus (DE-588)4308006-6 gnd Leichte Kette (DE-588)4502527-7 gnd Rekombinantes Protein (DE-588)4277353-2 gnd Antikörper (DE-588)4002290-0 gnd |
| topic_facet | Tomatenbronzefleckenvirus Leichte Kette Rekombinantes Protein Antikörper Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010046762&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT zhangmeiyun generationcharacterizationandexpressionofsinglechainantibodyfragmentsscfvsspecificforthensmmovementproteinoftomatospottedvirustswv |