Aus dem Veterinärwissenschaftlichen Department der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Arbeit angefertigt unter der Leitung von: Univ.-Prof. Dr. Gerd Sutter Angefertigt im Institut für Virologie des Helmholtz Zentrum München (apl.-Prof. Dr. Volker Bruss) In vitro Selection and Characterization of single stranded DNA Aptamers Inhibiting the Hepatitis B Virus Capsid-Envelope Interaction Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München von Ahmed El-Sayed Abd El-Halem Orabi aus Sharkia/Ägypten München 2013 Gedruckt mit der Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Joachim Braun Berichterstatter: Univ.-Prof. Dr. Gerd Sutter Korreferent: Univ.-Prof. Dr. Bernd Kaspers Tag der Promotion: 20. Juli 2013 My Family Contents Contents 1 INTRODUCTION ................................................................................................... 1 2 REVIEW OF THE LITERATURE ....................................................................... 2 2.1 HEPATITIS B VIRUS (HBV) .............................................................................................. 2 2.1.1 HISTORY AND TAXONOMY .......................................................................................................... 2 2.1.2 EPIDEMIOLOGY AND PATHOGENESIS .......................................................................................... 3 2.1.3 VIRION STRUCTURE ..................................................................................................................... 4 2.1.3.1 ROTEIN COMPOSITION OF HBV PARTICLES ............................................................................................... 4 2.1.3.1.1 SURFACE PROTEINS (HBS).................................................................................................................. 4 2.1.3.1.2 TRANSMEMBRANE TOPOLOGY OF SURFACE PROTEINS (HBS) ............................................................. 6 2.1.3.1.3 CORE PROTEIN (HBC) ......................................................................................................................... 7 2.1.3.1.4 HBE PROTEIN ....................................................................................................................................... 9 2.1.3.1.5 HEPTATITIS B POLYMERASE (P) PROTEIN ............................................................................................ 9 2.1.3.1.6 HEPTATITIS B X PROTEIN (HBX) ......................................................................................................... 9 2.1.3.2 HBV GENOME ........................................................................................................................................ 10 2.1.4 HBV LIFE CYCLE ....................................................................................................................... 11 2.1.5 ENVELOPMENT OF CORE PARTICLES .......................................................................................... 12 2.2 APTAMERS ....................................................................................................................... 14 2.2.1 NATURE AND THEORY ............................................................................................................... 14 2.2.2 TECHNOLOGY ............................................................................................................................ 14 2.2.2.1 OLIGONUCLEOTIDE LIBRARY ................................................................................................................... 14 2.2.2.2 STANDARD SELECTION PROCESS (SELEX) .............................................................................................. 16 2.2.2.3 SITE-DIRECTED SELECTION OF APTAMERS ............................................................................................... 18 2.2.2.4 AUTOMATED APTAMER SELECTION ......................................................................................................... 19 2.2.3 APTAMERS AND ANTIBODIES ..................................................................................................... 19 2.2.4 APTAMERS IN DIAGNOSTICS ...................................................................................................... 20 2.2.5 APTAMERS IN THERAPEUTICS .................................................................................................... 21 2.2.6 APTAMERS AGAINST HEPATITIS VIRUSES .................................................................................. 21 3 OBJECTIVES........................................................................................................ 22 4 MATERIAL AND METHODS ............................................................................ 23 4.1 MATERIAL ....................................................................................................................... 23 4.1.1 ANTIBODIES ............................................................................................................................. 23 4.1.2 APTAMERS ............................................................................................................................... 23 4.1.3 BACTERIAL STRAINS ................................................................................................................ 23 4.1.4 BACTERIAL MEDIA AND ANTIBIOTICS ...................................................................................... 24 4.1.5 CAPSIDS OF HBV ..................................................................................................................... 24 4.1.6 CELL LINE ................................................................................................................................ 24 4.1.7 CELL CULTURE MEDIA ............................................................................................................. 24 4.1.8 CHEMICALS AND REAGENTS .................................................................................................... 24 4.1.9 ENZYMES ................................................................................................................................. 26 4.1.9.1 RESTRICTION ENZYMES .......................................................................................................................... 26 4.1.9.2 OTHER ENZYMES .................................................................................................................................... 26 [III] Contents 4.1.10 DEVICES ................................................................................................................................... 26 4.1.11 KIT SYSTEMS ............................................................................................................................ 28 4.1.12 LABORATORY CONSUMABLES ................................................................................................. 28 4.1.13 DNA AND PROTEIN MARKERS ................................................................................................... 28 4.1.14 PLASMIDS ................................................................................................................................. 29 4.1.15 SOLUTIONS AND BUFFER SYSTEMS .......................................................................................... 32 4.1.16 PRIMERS ................................................................................................................................... 33 4.1.17 SOFTWARE ............................................................................................................................... 34 4.2 METHODS ........................................................................................................................ 35 4.2.1 DNA TECHNOLOGY .................................................................................................................. 35 4.2.1.1 CONVENTIONAL POLYMERASE CAHIN REACTION (PCR) ......................................................... 35 4.2.1.1.1 PCR DURING HBV WT AND MUTANT CAPSID EXPRESSION ................................................................. 35 4.2.1.1.2 PCR DURING APTAMER SELECTION AND SEQUENCING ......................................................................... 36 4.2.1.2 PURIFICATION AND CONCENTRATION OF DNA ......................................................................... 36 4.2.1.2.1 PHEENOL CHLOROFORM EXTRACTION .................................................................................................. 36 4.2.1.2.2 ETHANOL PRECIPITATION OF DNA ....................................................................................................... 37 4.2.1.2.3 PURIFICATION OF DNA SOLUTIONS AND PCR PRODUCTS ...................................................................... 37 4.2.1.3 GEL ELECTROPHORESIS ........................................................................................................... 37 4.2.1.3.1 AGAROSE GEL ELECTROPHORESIS ....................................................................................................... 37 4.2.1.3.2 DENATURING-UREA POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) ........................................... 37 4.2.1.4 EXTRACTION OF DNA FROM GELS ............................................................................................ 38 4.2.1.4.1 EXTRACTION FROM AGAROSE GEL .....................................................................................................