Institute of Virology University of Veterinary Medicine Hannover Isolation and Characterisation of High-Affinity Peptides Directed Against Surface Glycoproteins of Pathogenic Viruses THESIS submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY (Ph.D.) at the University of Veterinary Medicine Hannover by Carolin Hüttinger (Augsburg) Hannover 2009 Supervisor: Prof. Dr. Georg Herrler Advisory Committee: Prof. Dr. Georg Herrler Prof. Dr. Hassan Y. Naim Prof. Dr. Beate Sodeik 1st Evaluation: Prof. Dr. Georg Herrler (University of Veterinary Medicine Hannover, Institute of Virology) Prof. Dr. Hassan Y. Naim (University of Veterinary Medicine Hannover, Institute of Physiological Chemistry) Prof. Dr. Beate Sodeik (University of Medicine Hannover, Institute of Virology) 2nd Evaluation: Prof. Dr. Wolfgang Garten (Phillips University Marburg, Institute of Virology) Date of oral exam: 17.03.2009 Meiner Schwester - weil Agouti auch nur eine Fellfarbe ist - und meinen Eltern Table of contents Table of contents 1. Introduction…………………………………………………………………………………........ 1 1.1 Bacteriophage M13 ......................................................................................................1 1.1.1 Taxonomy of bacteriophage M13...........................................................................1 1.1.2 Virus structure of bacteriophage M13 ....................................................................2 1.1.3 Replication cycle of bacteriophage M13.................................................................3 1.1.4 Phage display ........................................................................................................4 1.2 Respiratory syncytial virus ............................................................................................5 1.2.1 Taxonomy of RSV .................................................................................................5 1.2.2 Virus structure of RSV ...........................................................................................7 1.2.3 Replication cycle of RSV........................................................................................8 1.2.4 Distribution and epidemiology of RSV....................................................................9 1.2.5 Clinical picture, prophylaxis and therapy of RSV infection ...................................10 1.2.6 Diagnostics of RSV..............................................................................................11 1.2.7 RSV fusion protein as a biopanning target...........................................................11 1.3 SARS coronavirus (CoV)............................................................................................13 1.3.1 Taxonomy of SARS-CoV .....................................................................................13 1.3.2 Structure of SARS-CoV .......................................................................................14 1.3.3 Replication cycle of SARS-CoV ...........................................................................16 1.3.4 Distribution and epidemiology of SARS-CoV .......................................................17 1.3.5 Clinical picture, prophylaxis and therapy of SARS-CoV .......................................18 1.3.7 Diagnostics of SARS-CoV ...................................................................................19 1.3.7 SARS-CoV spike protein as a biopanning target..................................................19 2. Aims of the study…………………………………………………………………... ................21 3. Materials……………………………………………………………………………...............….23 3.1 Cell lines....................................................................................................................23 3.2 Cell culture ................................................................................................................23 3.3 Viruses ......................................................................................................................24 3.4 Bacteria .....................................................................................................................24 3.5 Plasmids....................................................................................................................24 3.6 Synthetic genes.........................................................................................................25 3.7 Transfection reagent..................................................................................................25 3.8 Enzymes ...................................................................................................................25 Table of contents 3.9 Kits............................................................................................................................26 3.10 Primer .....................................................................................................................26 3.11 Peroxidase substrate...............................................................................................26 3.12 Antibodies ...............................................................................................................26 3.13 Marker SDS Page and agarose gel electrophoresis ................................................27 3.14 Chemicals ...............................................................................................................27 3.15 Media, buffers and solutions....................................................................................29 3.17 Synthetic peptides ...................................................................................................35 3.18 Equipment ...............................................................................................................36 3.19 Consumables ..........................................................................................................36 4. Methods……………………………………………………………………………….……… ….38 4.1 Cytological methods ..................................................................................................38 4.1.1 Culturing eukaryotic cells....................................................................................38 4.1.2 Transient transfection of eukaryotic cells ............................................................38 4.1.3 Concentration of cell culture supernatant............................................................39 4.2 Virological methods ...................................................................................................39 4.2.1 Virus amplification ..............................................................................................39 4.2.2 Virus titration ......................................................................................................40 4.2.3 Generation of VSV-pseudotypes ........................................................................41 4.2.4 Virus purification (via sucrose gradient or sucrose cushion)................................42 4.3 Phage display technique ...........................................................................................42 4.3.1 Target immobilisation..........................................................................................42 4.3.2 Biopanning .........................................................................................................44 4.3.3 Amplification .......................................................................................................44 4.3.4 Precipitation........................................................................................................45 4.3.5 Titration ..............................................................................................................45 4.3.6 Preparation of stocks ..........................................................................................45 4.4 Molecular biology ......................................................................................................46 4.4.1 DNA extraction from phages...............................................................................46 4.4.2 Polymerase chain reaction (PCR).......................................................................46 4.4.3 Agarose gel electrophoresis ...............................................................................47 4.4.4 DNA sequencing.................................................................................................47 4.4.5 Heat shock transformation..................................................................................47 4.4.6 Plasmid DNA preparation ...................................................................................48 Table of contents 4.5 Biochemical methods ................................................................................................48 4.5.1 BCA assay..........................................................................................................48 4.5.2 ELISA and Sandwich-ELISA...............................................................................49 4.5.3 Immunofluorescence analysis.............................................................................50 4.5.4 Cell lysis .............................................................................................................51 4.5.5 Co-immunoprecipitation......................................................................................51 4.5.6 Western blot and phage-overlay assay ...............................................................52