Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2003
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Leipzig, Univ., Diss., 2003 |
| Beschreibung: | 118 Bl. Ill., graph. Darst. |
Internformat
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Datensatz im Suchindex
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|---|---|
| adam_text |
CONTENTS
1. INTRODUCTION 1
1.2. BIOLOGICAL BACKGROUND 1
1.2. CYCLIC NUCLEOTIDE SIGNALING 2
1.3. THE CYCLIC NUCLEOTIDE PHOSPHODIESTERASE SUPERFAMILY 3
1.4. MOLECULAR BIOLOGY OF PDES 4
1.5. PHARMACOLOGY OF PDES 9
1.6. OBJECTIVES OF THE THESIS 13
2. MATERIALS
14
2.1. COMPETENT CELLS 14
2.2. CELL LINES 14
2.3. CLONING AND EXPRESSION VECTORS 14
2.4. ANTIBODIES 15
2.5. OLIGONUCLEOTIDES 15
2.5.1. UNIVERSAL PCR AND SEQUENCING PRIMERS 16
2.5.2. PRIMERS FOR PCR AMPLIFICATION AND SEQUENCING OF PDE5A1 16
2.5.3. PRIMERS FOR PCR AMPLIFICATION AND SEQUENCING OF PDE7B 17
2.5.4. PRIMERS FOR PCR AMPLIFICATION AND SEQUENCING OF PDE8B 17
2.5.5. PRIMERS FOR THE CRACE PROCEDURE
18
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/968456731
2.6. MOLECULAR BIOLOGICAL PRODUCTS 18
2.7. MEDIA AND BUFFER FORMULATIONS 19
2.7.1. MICROBIOLOGICAL MEDIA 19
2.7.2. CELL CULTURE MEDIA 20
2.8. CHEMICALS 21
2.9. INSTRUMENTS 23
2.10. SOFTWARE 24
3. METHODS
25
3.1. ISOLATION OF TOTAL-RNA FROM HUMAN TISSUES 25
3.2. QUANTIFICATION OF TOTAL RNA 25
3.3. RT-PCR (REVERSE TRANSCRIPTION - POLYMERASE CHAIN REACTION) 26
3.3.1. RT (CDNA SYNTHESIS) 26
3.3.2. POLYMERASE CHAIN REACTION (PCR) 26
3.4. CRACE 28
3.5. ANALYSIS OF DNA BY AGAROSE GEL ELECTROPHORESIS 29
3.6. ISOLATION OF DNA FRAGMENTS WITH MINELUTE
YY
PCR PURIFICATION KIT 30
3.7. PURIFICATION OF DNA FRAGMENTS FROM AGROSE GEL WITH
HIGH PURE PCR PRODUCT PURIFICATION KIT 31
3.8. TOPO TA CLONING
32
3.9. TRANSFORMATION INTO TOP 10
E. COLI 33
3.10. BLUE/WHITE SCREENING 33
3.11. MINI SCALE PLASMID ISOLATION (QIAPREP MINIPREP) 33
3.12. LARGE SCALE PLASMID ISOLATION (MAXIPREP) 35
3.13. QUANTIFICATION OF PLASMID DNA IN SOLUTION 37
3.14. RESTRICTION DIGESTION 37
3.15. LIGATION OF DNA FRAGMENTS WITH T4 DNA LIGASE 37
3.16. PRODUCTION OF RECOMBINANT PROTEIN IN
INSECT CELLS BY BAC-TO-BAC BACULOVIRUS EXPRESSION SYSTEM 38
3.16.1. CLONING INTO PFASTBAC DONOR PLASMID 38
3.16.2. TRANPOSITION 38
3.16.3. ISOLATION OF RECOMBINANT BACMID DNA 40
3.16.4. ANALYSIS OF RECOMBINANT BACMID DNA 41
3.16.5. TRANSFECTION OF SF9 CELLS WITH RECOMBINANT BACMID DNA 42
3.16.6. HARVEST/STORAGE OF RECOMBINANT BACULOVIRUS 43
3.16.7. INFECTION OF INSECT CELLS WITH RECOMBINANT BACULOVIRUS 43
3.17. EXPRESSION OF RECOMBINENT PDE TYPES IN COS1 CELLS 43
3.18. DETERMINATION OF PROTEIN CONCENTRATION 44
3.19. PREPARATION OF CELLULAR FRACTIONS (COS1 AND SF9 CELLS) 44
3.20. EN
2
YME ACTIVITY ASSAY
45
3.21. SDS - POLYACRYLAMIDE ELECTROPHORESIS (SDS-PAGE)
46
3.22. WESTERN BLOTTING 48
3.23. STAINING OF PROTEIN WITH COOMASSIE-BRILIANT BLUE 49
4. RESULTS 51
4.1. CLONING EXPRESSION AND CHARACTERIZATION OF HUMAN PDE5 A1 51
4.1.1 ISOLATION, CLONING AND SEQUENCE ANALYSIS OF A CDNA ENCODING HUMAN
PDE5A1 51
4.1.2. EXPRESSION OF RECOMBINANT HUMAN PDE5A1 IN COS1 CELLS 53
4.1.3. GENERATION OF RECOMBINANT BACULOVIRUS ENCODING HUMAN PDE5A1 55
4.1.4. CHARACTERIZATION OF RECOMBINANT HUMAN PDE5A1 EXPRESSED IN SF9
CELLS 57
4.2. CLONING EXPRESSION AND CHARACTERIZATION OF HUMAN PDE7B 59
4.2.1. AMPLIFICATION CLONING AND SEQUENCE ANALYSIS OF HUMAN PDE7B CDNA
59
4.2.2. RECOMBINANT EXPRESSION OF HUMAN PDE7B IN COS1 CELLS 60
4.2.3. GENERATION OF A RECOMBINANT BACULOVIRUS ENCODING HUMAN PDE7B 62
4.2.4. CHARACTERIZATION OF RECOMBINANT HUMAN PDE7B EXPRESSED IN SF9
CELLS 64
4.3. CLONING EXPRESSION AND CHARACTERIZATION OF HUMAN PDE8B 66
4.3.1. AMPLIFICATION CLONING AND SEQUENCE ANALYSIS OF A HUMAN PDE8B CDNA
66
4.3.2. GENERATION OF CDNA ENCODING THE FULL-LENGTH HUMAN PDE8B 69
4.3.3. EXPRESSION OF RECOMBINANT HUMAN PDE8B IN COS1 CELLS 72
4.3.4. GENERATION OF A RECOMBINANT BACULOVIRUS ENCODING HUMAN PDE8B 74
4.3.5. CHARACTERIZATION OF RECOMBINANT HUMAN PDE8B EXPRESSED IN SF9
CELLS 75
5. DISCUSSION
79
5.1. CLONING EXPRESSION AND CHARACTERIZATION OF HUMAN PDE5A1 79
5.1.1. GENERATION OF RECOMBINANT VECTORS ENCODING HUMAN PDE5A1 79
5.1.2. EXPRESSION OF HUMAN PDE5A1 IN COS1 AND SF9 CELLS 80
5.2. CLONING EXPRESSION AND CHARACTERIZATION OF HUMAN PDE7B 81
5.2.1. GENERATION OF RECOMBINANT VECTORS ENCODING HUMAN PDE7B 81
5.2.2. EXPRESSION OF HUMAN PDE7B IN COS1 AND SF9 CELLS 81
5.3. CLONING EXPRESSION AND CHARACTERIZATION OF HUMAN PDE8B 82
5.3.1. GENERATION OF RECOMBINANT VECTORS ENCODING HUMAN PDE8B 82
5.3.2. EXPRESSION OF HUMAN PDE8B IN COS1 AND SF9 CELLS 85
6. SUMMARY
87
7. REFERENCES
91
8. APPENDIX
104 |
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| author | Unciuleac, Liviu 1968- |
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| bvnumber | BV017525014 |
| ctrlnum | (OCoLC)634330979 (DE-599)BVBBV017525014 |
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| genre_facet | Hochschulschrift |
| id | DE-604.BV017525014 |
| illustrated | Illustrated |
| indexdate | 2024-11-22T17:38:16Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-010556688 |
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| physical | 118 Bl. Ill., graph. Darst. |
| publishDate | 2003 |
| publishDateSearch | 2003 |
| publishDateSort | 2003 |
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| spelling | Unciuleac, Liviu 1968- Verfasser (DE-588)12487262X aut Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs vorgelegt von Liviu Unciuleac 2003 118 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Leipzig, Univ., Diss., 2003 Phosphodiesterasen (DE-588)4174389-1 gnd rswk-swf Antiasthmatikum (DE-588)4201239-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Antiasthmatikum (DE-588)4201239-9 s Phosphodiesterasen (DE-588)4174389-1 s b DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010556688&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Unciuleac, Liviu 1968- Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs Phosphodiesterasen (DE-588)4174389-1 gnd Antiasthmatikum (DE-588)4201239-9 gnd |
| subject_GND | (DE-588)4174389-1 (DE-588)4201239-9 (DE-588)4113937-9 |
| title | Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs |
| title_auth | Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs |
| title_exact_search | Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs |
| title_full | Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs vorgelegt von Liviu Unciuleac |
| title_fullStr | Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs vorgelegt von Liviu Unciuleac |
| title_full_unstemmed | Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs vorgelegt von Liviu Unciuleac |
| title_short | Cloning, expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs |
| title_sort | cloning expression and characterization of cyclic nucleotide phosphodiesterases as potential targets for antiasthmatic drugs |
| topic | Phosphodiesterasen (DE-588)4174389-1 gnd Antiasthmatikum (DE-588)4201239-9 gnd |
| topic_facet | Phosphodiesterasen Antiasthmatikum Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010556688&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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