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Das Komplementsystem Die Bedeutung verschiedener CRASP-Proteine für die Komplementresistenz von Borrelia burgdorferi s.s. Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biowissenschaften der Johann Wolfgang Goethe - Universität in Frankfurt am Main von Corinna Siegel aus Sebnitz Frankfurt 2010 (D 30) vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe - Universität als Dissertation angenommen. Dekan: Prof. Dr. A. Starzinski-Powitz Gutachter: Prof. Dr. V. Müller Prof. Dr. P. Kraiczy Datum der Disputation: Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis ..................................................................................................... I Abkürzungsverzeichnis ........................................................................................ VIII I. Einleitung ........................................................................................................... 1 1 Die Multisystemkrankheit Lyme-Borreliose ........................................................... 1 2 Der Überträger Ixodes spp. ................................................................................... 2 3 Charakteristika des Erregers B. burgdorferi s.l. .................................................... 3 3. 1 Taxonomie und Morphologie ......................................................................... 4 3. 2 Das Genom ................................................................................................... 6 3. 3 Genetische Manipulation von B. burgdorferi s.l. ............................................ 7 4 Das humane Immunsystem .................................................................................. 7 4. 1 Das Komplementsystem ................................................................................ 7 4. 2 Die Faktor H-Proteinfamilie ......................................................................... 10 4. 2. 1 Faktor H und FHL-1 ............................................................................. 11 4. 2. 2 Faktor H verwandte Proteine (CFHRs) ................................................ 13 5 Komplementevasion von pathogenen Mikroorganismen .................................... 14 5. 1 Komplementevasionsstrategien von B. burgdorferi s.l. ................................ 16 5. 2 Eigenschaften von CRASP der humanpathogenen Spezies B. burgdorferi s.s. ........................................................................................ 17 6 Fragestellung und Zielsetzung ............................................................................ 21 II. Material und Methoden ....................................................................................22 1 Organismen, Vektoren, Oligonukleotide, Antikörper ........................................... 22 1.1 Organismen .................................................................................................. 22 1.2 Vektoren ....................................................................................................... 22 1.3 Starteroligonukleotide ................................................................................... 24 2 Antikörper, rekombinante Proteine, Humanserum .............................................. 26 2.1 Antikörper ..................................................................................................... 26 2.2 Rekombinante Proteine ................................................................................ 27 2.3 Humanserum ................................................................................................ 27 3 Kulturmedien ....................................................................................................... 28 3.1 Kulturmedium für B. burgdorferi s.l. .............................................................. 28 3.2 Kulturmedium für E. coli ............................................................................... 29 I Inhaltsverzeichnis 4 Kultivierung von Bakterien .................................................................................. 30 4.1 Kultivierung von B. burgdorferi s.l. ................................................................ 30 4.2 Bestimmung der Zellkonzentration von B. burgdorferi s.l. ............................ 31 4.3 Kultivierung von E. coli ................................................................................. 31 4.4 Bestimmung der Zelldichte von E. coli .......................................................... 31 5 Molekularbiologische Methoden .......................................................................... 32 5.1 Isolierung von Plasmid-DNA aus B. burgdorferi s.l. ...................................... 32 5.2 Plasmidisolierung aus E. coli ........................................................................ 33 5.3 Photometrische DNA-Konzentrationsbestimmung ....................................... 33 5.4 Aufkonzentration von Plasmid-DNA ............................................................. 33 5.5 Polymerasekettenreaktion (PCR) ................................................................. 33 5.6 PCR aus einer Bakterienflüssigkultur ........................................................... 34 5.7 Gelelektrophorese ........................................................................................ 35 5.7.1 Agarose-Gelelektrophorese ................................................................... 35 5.7.2 Pulsfeld-Gelelektrophorese (PFGE) ....................................................... 35 5.8 Aufreinigung von PCR-Fragmenten.............................................................. 36 5.9 In vitro-Mutagenese ...................................................................................... 36 5.10 Transformation von Bakterien mit Plasmid-DNA ........................................ 37 5.10.1 Elektroporation von B. garinii G1 ......................................................... 37 5.10.2 Transformation von E. coli DH5-Zellen mittels CaCl2-Hitzeschock .... 38 5.10.2.1 Herstellung kompetenter E. coli DH5-Zellen ............................... 38 5.10.2.2 CaCl2-Hitzeschock-Methode ......................................................... 38 5.11 Southernblot-Analyse ................................................................................. 39 5.11.1 DNA-Transfer auf Nylonmembranen mittels Vakuum .......................... 39 5.11.2 Hybridisierung immobilisierter DNA mit HRP-markierten Gensonden .......................................................................................... 40 5.12 Sequenzierung von Plasmid-DNA .............................................................. 40 6 Proteinbiochemische Methoden .......................................................................... 41 6.1 Herstellung von Zellextrakten von B. burgdorferi s.l. .................................... 41 6.2 Denaturierende Polyacrylamidgelelektrophorese (SDS-PAGE) ................... 41 6.2.1 Separation von Proteinen in der Tris Tricin-SDS-Gelelektrophorse ....... 41 6.2.2 Separation von Proteinen nach Laemmli ............................................... 42 6.3 Westernblot- und Ligandenaffinitätsblot-Analyse ......................................... 42 II Inhaltsverzeichnis 6.4 Silberfärbung von SDS-Polyacrylamidgelen ................................................. 43 6.5 Kolloidale Coomassie-Färbung von SDS-Polyacrylamidgelen ..................... 44 6.6 Massenspektrometrie ................................................................................... 45 6.7 Produktion rekombinanter Proteine in E. coli ................................................ 45 6.8 Affinitätschromatographische Aufreinigung rekombinanter Proteine ............ 46 6.9 Konzentrationsbestimmung rekombinanter Proteine .................................... 47 7 Immunologische Methoden ................................................................................. 47 7.1 Hämolytischer Assay .................................................................................... 47 7.2 Immunfluoreszenzmikroskopie ..................................................................... 48 8 Spezielle Methoden ............................................................................................ 49 8.1 Serumadsorption mittels magnetischer Partikel ........................................... 49 8.2 Serumadsorption .......................................................................................... 50 8.3 Kofaktor-Assay ............................................................................................. 52 8.4 Wachstums-Inhibitions-Assay ...................................................................... 53 8.5 Protease-Assay ............................................................................................ 54 9 Chemikalien ........................................................................................................ 55 III. Ergebnisse ........................................................................................................56 1 Transformation von B. garinii G1 ........................................................................ 56 1.1 Herstellung von Vektorkonstrukten mit mutagenisierten cspA-Genen .......... 57 1.2 Charakterisierung der verschiedenen CRASP-produzierenden Transformanten von B. garinii G1 ................................................................. 58 1.3 Charakterisierung der Transformanten hinsichtlich ihres Plasmidprofils und genetische Lokalisation der CRASP-kodierenden Gene ....................... 62 1.4 Nachweis der CRASP-Produktion
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