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Dissertation Christina Röser

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Dissertation Christina Röser Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) Untersuchungen zum Replikationsverhalten des Hepatitis C Virus (HCV) in Zellkultursystemen mit manipuliertem 2’- 5’Oligoadenylatsynthetase-System vorgelegt von Christina Röser geboren in Erfurt/Thüringen Februar 2007 Eingereicht im Fachbereich 2 (Biologie/Chemie) der Universität Bremen Erster Gutachter: PD Dr. rer. nat. Andreas Dotzauer Universität Bremen, Fachbereich 2 Zweiter Gutachter: Prof. Dr. Reimer Stick Universität Bremen, Fachbereich 2 Tag des öffentlichen Kolloquiums: 19. April 2007 (Universität Bremen) Inhaltsverzeichnis 1. Einleitung.............................................................................................. 1 1.1. Hepatitis C Virus .................................................................................. 1 1.1.1. Taxonomie............................................................................................ 1 1.1.2. Genomorganisation............................................................................... 2 1.1.3. Genotypen............................................................................................. 3 1.1.4. Verbreitung........................................................................................... 5 1.1.5. Replikation............................................................................................ 5 1.1.6. Übertragungswege ................................................................................ 7 1.1.7. Krankheitsbild und Therapie ................................................................ 7 1.1.8. Diagnostik............................................................................................. 9 1.1.8.1. Serologischer Antikörper Test.............................................................. 9 1.1.8.2. Virologische PCR Tests zum Nachweis von HCV RNA................... 10 1.1.9. Hepatitis C Virus in Zellkultur ........................................................... 11 1.2. Unspezifische Immunabwehr ............................................................. 13 1.2.1. 2’- 5’Oligoadenylatsynthetase-System und RNase L......................... 14 1.2.2. RNase L Inhibitor (RLI) ..................................................................... 16 1.3. Polymerase Kettenreaktion................................................................. 17 1.3.1. Historisches ........................................................................................ 17 1.3.2. Prinzip der PCR.................................................................................. 18 1.3.3. Real time PCR .................................................................................... 19 1.3.4. Fluoreszenzfarbstoffe ......................................................................... 20 1.3.5. ABI PRISM™ Systeme...................................................................... 21 1.3.6. RotorGene™....................................................................................... 22 1.4. Ziel der Arbeit .................................................................................... 24 2. Material und Methoden ...................................................................... 25 2.1. Materialien.......................................................................................... 25 2.1.1. Antikörper........................................................................................... 25 2.1.2. Bioreagenzien ..................................................................................... 25 2.1.3. Gebrauchsfertige Kits......................................................................... 26 2.1.4. Geräte.................................................................................................. 26 2.1.5. Medien und Lösungen ........................................................................ 27 2.1.6. Oligonukleotide .................................................................................. 27 2.1.7. Plasmide.............................................................................................. 29 2.1.8. Restriktionsendonukleasen ................................................................. 30 2.1.9. Software.............................................................................................. 30 2.1.10. Verbrauchsmaterialien........................................................................ 31 2.1.11. virologische Materialien ..................................................................... 31 2.1.12. Zelllinien............................................................................................. 31 2.2. Methoden ............................................................................................ 32 2.2.1. Molekularbiologie .............................................................................. 32 2.2.1.1. Aufreinigung von Zell-RNA mit dem RNeasy® Mini Kit.................. 32 2.2.1.2. DNA-Auftrennung im Agarosegel ..................................................... 32 2.2.1.3. cDNA-Synthese .................................................................................. 32 2.2.1.4. Real time PCR zum quantitativen Nachweis der mRNA von RNase L, RLI und GAPDH sowie der 18S rRNA ..................... 33 2.2.1.5. Full length Amplifikation des RNase L Inhibitor .............................. 34 2.2.1.6. Klonierung des RLI in pcDNA3.1/V5-His und Transformation von E.coli............................................................................................ 35 2.2.1.7. Klonscreening mittels PCR ................................................................ 36 2.2.1.8. Präparation der rekombinanten Plasmide........................................... 37 2.2.1.9. Sequenzierung .................................................................................... 37 2.2.1.10. Umklonierung des RLI aus pcDNA3.1/V5-His_RLI in pl.18............ 37 2.2.1.11. In vitro Transkription von HCV full length HCV RNA..................... 39 2.2.1.12. Entwicklung eines HCV RotorGene Nachweissystems ..................... 40 2.2.2. Zellkultur und virologische Methoden ............................................... 42 2.2.2.1. Kryokonservierung von Huh7- und FRhK-4-Zellen .......................... 42 2.2.2.2. Auftauen von Huh7- und FRhK-4-Zellen .......................................... 42 2.2.2.3. Passagieren von Huh7- und FRhK-4-Zellen ...................................... 42 2.2.2.4. Transfektion mit FuGene 6 Transfection Reagent ............................. 43 2.2.2.5. Transfektion mit jetPEI ...................................................................... 43 2.2.2.6. G418 Titration für Huh7-Zellen ......................................................... 43 2.2.2.7. Stabile Überexpression des RLI in Huh7- und FRhK-4-Zellen ......... 44 2.2.2.8. Immunfluoreszenz .............................................................................. 44 2.2.2.9. Stabilitätstest von HCV RNA in zellfreiem Medium......................... 45 2.2.2.10. HCV Infektion von FRhK-4-RLI-Klonen .......................................... 45 2.2.2.11. HCV Infektion transient RLI-transfizierter Huh7- und FRhK-4-Zellen............................................................................. 46 2.2.2.12. Transfektion in vitro transkribierter RNA in Huh7- und FRhK-4-Zellen............................................................................. 46 2.2.2.13. Transfektion in vitro transkribierter HCV full length RNA in transient RLI-transfizierten Huh7- und FRhK-4-Zellen..................... 47 2.2.2.14. Aufreinigung viraler RNA mit dem QIAamp®Viral RNA Mini Kit.. 47 3. Ergebnisse........................................................................................... 48 3.1. Erstellung der real time PCRs ............................................................ 48 3.1.1. Real time PCR zum quantitativen Nachweis der mRNA von RNase L, RLI und GAPDH sowie der 18S rRNA .................... 48 3.1.1.1. Effizienz.............................................................................................. 49 3.1.1.2. Spezifität............................................................................................. 51 3.1.2. Real time RT-PCR zum quantitativen Nachweis der HCV RNA ...... 52 3.1.2.1. Testung des HCV RotorGene Assay auf Kreuzreaktivität ................. 52 3.1.2.2. Sensitivität des HCV RotorGene Nachweissystems .......................... 53 3.1.2.3. Nachweis verschiedener HCV Subtypen............................................ 55 3.1.3. Zusammenfassung real time PCRs..................................................... 57 3.2. RNase L Inhibitor ............................................................................... 57 3.2.1. Sequenzierung RNase L Inhibitor ...................................................... 57 3.2.2. Transfektion mit jetPEI bzw. FuGene 6 Transfection Reagent.......... 59 3.2.3. RLI-Kinetik nach Transfektion mit pcDNA3.1/V5-His_RLI ............ 61 3.2.4. Vergleich der Expressionsraten von pcDNA3.1/V5-His_RLI und pl.18_RLI..................................................................................... 62 3.2.5. Nachweis der RLI-Expression mittels Immunfluoreszenz................. 64 3.2.6. Stabile Überexpression des RLI in FRhK-4- und Huh7-Zellen ......... 65 3.2.7. RLI-Kinetik der FRhK-4-RLI-Klone 4 und 5 .................................... 67 3.2.8. Kinetik der RLI-Expression stabil transfizierter FRhK-4-6er Klone . 68 3.2.9. Zusammenfassung RNase L Inhibitor ...............................................
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