Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Volltext Inhaltsverzeichnis |
| Physical Description: | 122 Bl. graph. Darst. |
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MARC
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| 100 | 1 | |a Seo, Ean-Jeong |d 1979- |e Verfasser |0 (DE-588)1024499715 |4 aut | |
| 245 | 1 | 0 | |a Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins |c von Ean-jeong Seo |
| 246 | 1 | 3 | |a Konstruktion von rekombinanten E. coli Nissle 1917 (EcN) Stämmen, die Defensine exprimieren bzw. sekretieren |
| 264 | 1 | |c 2012 | |
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| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Würzburg, Univ., Diss., 2012 | ||
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Record in the Search Index
| _version_ | 1804149359523135488 |
|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENTS
SUMMARY L
ZUSAMMENFASSUNG 2
1. INTRODUCTION 4
1.1. PROBIOTIC ESCHERICHIA COLI STRAIN NISSLE 1917 (ECN) 4
1.1.1. PROBIOTICS 4
1.1.2. E. COLI NISSLE 1917 (ECN) 4
1.1.2.1. GENERAL PROPERTIES OF ECN 5
1.1.2.2. FITNESS FACTORS OF ECN 6
1.2. EXPRESSION OF ANTIMICROBIAL PEPTIDES IN THE GASTROINTESTINAL (GL)
TRACT 8
1.2.1. MICROBIAL/HOST INTERACTIONS 8
1.2.2. INTESTINAL EPITHELIAL CELLS AND THE EXPRESSION AND REGULATION OF
ANTIMICROBIAL PEPTIDES 9
1.2.3. ANTIMICROBIAL PEPTIDES 10
1.2.3.1. DEFENSINS 10
1.2.3.1.1. THE MECHANISM OF DEFENSINS 11
1.2.3.1.2. CLASSES OF DEFENSINS 11
1.2.3.1.2.1. HUMAN A-DEFENSINS 13
1.2.3.1.2.2. HUMAN B-DEFENSINS 13
1.3. INFLAMMATORY BOWEL DISEASES (IBD) 14
1.3.1. DEFENSIN DEFICIENCY IN INFLAMMATORY BOWEL DISEASES 15
1.3.1.1. THE ALPHA DEFENSIN DEFICIENCY IN CROHN S DISEASE 15
1.3.1.2. THE BETA-DEFENSIN DEFICIENCY IN CROHN S DISEASE 16
1.3.1.3. BETA DEFENSINS DEFICIENCY IN ULCERATIVE COLITIS 17
1.4. AIMS OF THIS THESIS 19
2. MATERIALS 21
2.1. BACTERIAL STRAINS 21
HTTP://D-NB.INFO/1024866572
IMAGE 2
2.2. PLASMIDS 23
2.3. OLIGONUCLEOTIDES 26
2.4. CHEMICALS, ENZYMES AND ANTIBODIES 28
2.5. TECHNICAL EQUIPMENTS 29
2.6. MEDIA 31
2.7. ANTIBIOTICS 32
2.8. ENZYMES AND SOLUTIONS 33
2.9. DNA AND PROTEIN MARKERS 33
2.9.1. DNA MARKER 33
2.9.2. PROTEIN MARKER 34
3. METHODS 35
3.1. NUCLEIC ACID METHODS 35
3.1.1. DESIGN OF HBD2 AND HD5 GENES WITH OPTIMIZED CODON USAGE FOR
EXPRESSION IN ESCHERICHIA COLI 35
3.1.2. PLASMID DNA EXTRACTIONS 35
3.1.2.1. RAPID METHOD FOR THE ISOLATION OF PLASMID DNA 35
3.1.2.2. PLASMID ISOLATION WITH PLASMID MINI KIT, QIAGEN 36
3.1.3. ENZYMATIC DIGEST OF DNA WITH RESTRICTION ENDONUCLEASES 37
3.1.4. HORIZONTAL GEL ELECTROPHORESIS 37
3.1.5. ISOLATION OF DNA FRAGMENTS FROM AGAROSE GELS 38
3.1.6. POLYMERASE CHAIN REACTION (PCR) 38
3.1.7. DEPHOSPHORYLATION OF DNA FRAGMENTS 39
3.1.8. LIGATION OF DNA FRAGMENTS 39
3.1.9. TRANSFORMATION OF BACTERIAL CELLS 39
3.1.9.1. PREPARATION OF ELECTROCOMPETENT CELLS 39
3.1.9.2. TRANSFORMATION OF BACTERIAL CELLS BY ELECTROPORATION 40
3.1.10. SEQUENCING WITH FLUORESCENT LABELED NUCLEOTIDES (AMPLITAQ FS
BIGDYE-TERMINATOR, PERKIN ELMER) 40
IMAGE 3
3.2. PROTEIN METHODS 41
3.2.1. PROTEIN EXPRESSION WITH PUREXPRESS IN VITRO PROTEIN SYNTHESIS
KIT (NEB) 41
3.2.2. PROTEIN EXPRESSION IN RECOMBINANT STRAINS 43
3.2.3. CELL LYSIS BY SONICATION 44
3.2.4. ISOLATION OF SOLUBLE FUSION PROTEIN WITH AN N TERMINAL HIS TAG BY
NICKEL AFFINITY CHROMATOGRAPHY 44
3.2.5. SDS TRICINE WESTERN BLOT ANALYSIS 45
3.2.5.1. TRICINE SDS-PAGE 45
3.2.5.2. WESTERN BLOTTING 47
3.2.5.3. ANTIBODY TREATMENT 48
3.2.6. ANTIMICROBIAL ACTIVITY TESTS 49
3.2.6.1. RADIAL DIFFUSION ASSAY 49
3.2.6.2. THE KILLING ASSAY IN LIQUID CULTURE 50
4. RESULTS 51
4.1. CODON OPTIMIZATION OF HUMAN BETA-DEFENSIN 2 AND HUMAN
ALPHA-DEFENSIN 5 GENE FOR EXPRESSION IN ESCHERICHIA COLI 51
4.2. CONSTRUCTION OF RECOMBINANT PLASMIDS FOR THE EXPRESSION OF
DEFENSINS IN E. COLI 53
4.2.1. CLONING OF NHD5-GER E IN PMUT1-TC UNDER THE CONTROL OF VARIOUS
PROMOTERS 53
4.2.2. EXPRESSION OF HD5 IN ECN AND ECNC AFTER CLONING INTO PMUT1-TC
UNDER THE CONTROL OF VARIOUS PROMOTERS 56
4.2.3. CLONING OF NHD5 GENE INTO LOW-COPY NUMBER EXPRESSION VECTORS FOR
EXPRESSION BY THE LACTOSE PROMOTER/OPERATOR SYSTEM 57
4.2.4. EXPRESSION OF HD5 IN E. COLI K-12 BL21 AFTER CLONING INTO
LOW-COPY NUMBER EXPRESSION VECTORS UNDER THE CONTROL OF THE LACTOSE
PROMOTER/OPERATOR SYSTEM 57
4.2.5. IN VITRO EXPRESSION OF NHD5 GENE 58
4.2.6. EXPRESSION OF NHD5 GENE AFTER CLONING INTO PT7-VECTORS UNDER
CONTROL OF THE T7-RNA POLYMERASE SPECIFIC PHI 10 PROMOTOR 59
4.2.7. EXPRESSION OF HD5 AND HBD2 FUSION PROTEINS IN ECN AND ECNC AFTER
CLONING INTO PET-28A(+) VECTOR CARRYING A HIS TAG AND UNDER THE CONTROL
OF THE T7-RNA POLYMERASE 60
IMAGE 4
4.3. EXPRESSION OF HBD2 AND HD5 FUSION PROTEINS IN ESCHERICHIA COLI 65
4.4. ISOLATION BY NIKEL-AFFINITY CHROMATOGRAPHY AND ANTIMICROBIAL
ACTIVITY OF HISMHBD2 68
4.5. SECRETION OF A MHBD2 FUSION PROTEIN BY RECOMBINANT E. COLI STRAINS
72
4.6. ANTIMICROBIAL ACTIVITY OF THE SECRETED FUSION PROTEIN YEBFMHBD2 77
5. DISCUSSION 83
5.1. CONSTRUCTION AND EXPRESSION OF CATIONIC ANTIMICROBIAL PEPTIDES,
I.E. DEFENSINS IN E. COLI 83
5.2. ANTIMICROBIAL ACTIVITY OF EXPRESSED FUSION MATURE HBD2 (HISMHBD2)
90
5.3. SECRETION OF A MHBD2 FUSION PROTEIN BY RECOMBINANT E. COLI STRAINS
91
5.4. ANTIMICROBIAL ACTIVITY OF SECRETED YEBF-MATURE HBD2 FUSION PROTEIN
(YEBFMHBD2) 92
5.5. CONCLUDING REMARKS 93
REFERENCES 95
SUPPLEMENTARY RESULTS 108
ABBREVIATIONS 113
PHYSICAL MAPS OF EXPRESSION PLASMIDS 116
SEQUENCES 119
PUBLICATION 120
PRESENTATIONS 120
CURRICULUM VITAE 121
|
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| language | English |
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| spelling | Seo, Ean-Jeong 1979- Verfasser (DE-588)1024499715 aut Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins von Ean-jeong Seo Konstruktion von rekombinanten E. coli Nissle 1917 (EcN) Stämmen, die Defensine exprimieren bzw. sekretieren 2012 122 Bl. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Würzburg, Univ., Diss., 2012 Defensine (DE-588)4836105-7 gnd rswk-swf Genexpression (DE-588)4020136-3 gnd rswk-swf Probiotikum (DE-588)4338112-1 gnd rswk-swf Rekombinante DNS (DE-588)4225578-8 gnd rswk-swf Escherichia coli (DE-588)4070959-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Escherichia coli (DE-588)4070959-0 s Probiotikum (DE-588)4338112-1 s Rekombinante DNS (DE-588)4225578-8 s Genexpression (DE-588)4020136-3 s Defensine (DE-588)4836105-7 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-72005 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-72005 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025183105&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Seo, Ean-Jeong 1979- Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins Defensine (DE-588)4836105-7 gnd Genexpression (DE-588)4020136-3 gnd Probiotikum (DE-588)4338112-1 gnd Rekombinante DNS (DE-588)4225578-8 gnd Escherichia coli (DE-588)4070959-0 gnd |
| subject_GND | (DE-588)4836105-7 (DE-588)4020136-3 (DE-588)4338112-1 (DE-588)4225578-8 (DE-588)4070959-0 (DE-588)4113937-9 |
| title | Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins |
| title_alt | Konstruktion von rekombinanten E. coli Nissle 1917 (EcN) Stämmen, die Defensine exprimieren bzw. sekretieren |
| title_auth | Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins |
| title_exact_search | Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins |
| title_full | Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins von Ean-jeong Seo |
| title_fullStr | Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins von Ean-jeong Seo |
| title_full_unstemmed | Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins von Ean-jeong Seo |
| title_short | Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins |
| title_sort | construction of recombinant e coli nissle 1917 ecn strains for the expression and secretion of defensins |
| topic | Defensine (DE-588)4836105-7 gnd Genexpression (DE-588)4020136-3 gnd Probiotikum (DE-588)4338112-1 gnd Rekombinante DNS (DE-588)4225578-8 gnd Escherichia coli (DE-588)4070959-0 gnd |
| topic_facet | Defensine Genexpression Probiotikum Rekombinante DNS Escherichia coli Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-72005 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025183105&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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