Development of bispecific filamentous bacteriophages for the generation of a novel automated screening system based on phage display technology [Elektronische Ressource] / vorgelegt von Tim Oliver Stolle

Stolle

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Development of bispecific filamentous bacteriophages for
the generation of a novel automated screening system
based on phage display technology



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation


vorgelegt von

Diplom-Biologe
Tim Oliver Stolle
aus
Mönchengladbach




Berichter: Universitätsprofessor Dr. rer. nat. R. Fischer
Universitätsprofessor Dr. rer. nat. F. Kreuzaler


Tag der mündlichen Prüfung: 14. März 2005

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Content I
I INTRODUCTION ............................................................................................................................ 1
I.1 FILAMENTOUS BACTERIOPHAGES.................................................................................... 2
I.1.1 Structure of filamentous bacteriophages........................................................................... 2
I.1.2 Genome of filamentous bacteriophages............................................................................ 5
I.1.3 Life cycle of filamentous bacteriophages .......................................................................... 8
I.1.3.1 Infection process of filamentous bacteriophages.......................................................... 8
I.1.3.2 Replication and translation of the phage genome....................................................... 10
I.1.3.3 Assembly process of filamentous bacteriophages...................................................... 12
I.2 PHAGE DISPLAY........................................................................................................... 13
I.3 RESEARCH OBJECTIVES............................................................................................... 17
I.3.1 Protein Nanochip ............................................................................................................. 17
I.3.2 Aim of this thesis.19
I.3.2.1 Generation of bispecific filamentous bacteriophages ................................................. 19
I.3.2.2 Immobilization of filamentous bacteriophages ............................................................ 21
II MATERIALS AND METHODS..................................................................................................... 23
II.1 MATERIALS.................................................................................................................. 23
II.1.1 Chemicals and consumables........................................................................................... 23
II.1.2 Enzymes and reaction kits............................................................................................... 23
II.1.3 Primary and secondary antibodies .................................................................................. 24
II.1.4 Vectors............................................................................................................................. 25
II.1.4.1 Phage vectors ............................................................................................................. 25
II.1.4.2 Phagemid vectors........................................................................................................ 25
II.1.5 Biological material............................................................................................................ 26
II.1.5.1 Bacterial strains........................................................................................................... 26
II.1.5.2 Helperphage................................................................................................................ 26
II.1.5.3 L540Cy cell line 26
II.1.6 Buffer, media and solutions ............................................................................................. 27
II.1.7 Oligonucleotides .............................................................................................................. 27
II.1.7.1 Oligonucleotides for construction and analysis of fd-g6m........................................... 27
II.1.7.2 for constructiis of fd-g6/9m........................................ 28
II.1.7.3 Oligonucleotides for amplification of CD30R cDNA .................................................... 28
II.1.7.4 for amplification of Ki-4(scFv) DNA ................................................. 28
II.1.7.5 Oligonucleotides for construction and analysis of pHEN9 .......................................... 29
II.1.7.6 for construction of intermediate pHEN9-Bi...................................... 29
II.1.7.7 Oligonucleotides for construction and analysis of pHEN3+9 29
II.1.7.8 for sequencing of pHEN3CWP+9.................................................... 29
II.1.7.9 Oligonucleotides for construction and analysis of pHEN3+9Strep ............................. 30
II.1.7.10 Oligonucleotides for construction and analysis of pHEN9-Strep ............................... 30
II.1.8 Equipment........................................................................................................................ 30 Content II
II.1.9 Approbation for the conducted work................................................................................ 31
II.2 METHODS.................................................................................................................... 32
II.2.1 Recombinant DNA techniques ........................................................................................ 32
II.2.1.1 Isolation of plasmid, phage and phagemid DNA from E. coli...................................... 32
II.2.1.2 PCR amplification........................................................................................................ 32
II.2.1.3 Analytical agarose gel electrophoresis........................................................................ 35
II.2.1.4 Preparative gel electrophoresis................................................................................... 35
II.2.1.5 Quantification of nucleic acids..................................................................................... 35
II.2.1.6 Restriction digest of DNA ............................................................................................ 36
II.2.1.7 Ligation of DNA ........................................................................................................... 36
II.2.1.8 Growth and maintenance of bacterial strains.............................................................. 36
II.2.1.9 Bacterial transformation .............................................................................................. 36
II.2.1.10 DNA sequencing and sequencing analysis ............................................................... 37
II.2.1.11 RNA isolation and synthesis of first-strand cDNA...................................................... 38
II.2.2 Methods for phage analysis............................................................................................. 39
II.2.2.1 Phage amplification..................................................................................................... 39
II.2.2.2 Phage preparation....................................................................................................... 39
II.2.2.3 Phage titration ............................................................................................................. 40
II.2.2.4 Purification of recombinant phages............................................................................. 40
II.2.2.5 Phage ELISA............................................................................................................... 42
II.2.2.6 Electron microscopy.................................................................................................... 44
III RESULTS AND DISCUSSION 47
III.1 CONSTRUCTION AND ANALYSIS OF BISPECIFIC PHAGE VECTORS .................................... 47
III.1.1 Construction of bispecific phage vector fd-g6/9m ........................................................... 49
III.1.2 Analysis of bispecific phage vector fd-g6/9m .................................................................. 51
III.1.3 Construction and analysis of phage vector fd-g9m ......................................................... 55
III.1.3.1 Construction of phage vector fd-g9m.......................................................................... 55
III.1.3.2 Analysis of phage vector fd-g9m................................................................................. 56
III.2 CONSTRUCTION AND ANALYSIS OF BISPECIFIC PHAGEMIDS ............................................ 60
III.2.1 Construction and analysis of phagemid vector pHEN9................................................... 61
III.2.1.1 Construction of phagemid vector pHEN9.................................................................... 61
III.2.1.2 Analysis of phagemid vector pHEN9........................................................................... 62
III.2.2 Construction of bispecific phagemid vector pHEN3+9 .................................................... 64
III.2.3 Analysis of bispecific phagemid vector pHEN3+9...................................