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Untersuchungen Zum Nachweis Von Pflanzenviren Mit Peptiden Und Antibody Mimics Aus Phagenbibliotheken

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Untersuchungen Zum Nachweis Von Pflanzenviren Mit Peptiden Und Antibody Mimics Aus Phagenbibliotheken Untersuchungen zum Nachweis von Pflanzenviren mit Peptiden und Antibody Mimics aus Phagenbibliotheken Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades DOKTOR DER NATURWISSENSCHAFTEN Dr. rer. nat. genehmigte Dissertation von M. Sc. Dominik Lars Klinkenbuß geboren am 08.06.1986 in Dorsten 2016 Referent: Prof. Dr. Edgar Maiß Korreferent: Prof. Dr. Bernhard Huchzermeyer Tag der Promotion: 09.03.2016 KURZFASSUNG III KURZFASSUNG Trotz der kontinuierlichen Entwicklung neuerer und scheinbar fortschrittlicherer Methoden und Techniken für die Erkennung und Identifizierung von Pflanzenviren, eignen sich nur wenige dieser Methoden für Routinetests in Laboratorien. Aufgrund einzigartiger Merkmale, wie zum Beispiel die robuste Funktionalität bei einer genauen Reproduzierbarkeit, sind bis heute der enzyme-linked immunosorbent assay (ELISA) und die real-time polymerase chain reaction (qPCR) zwei der meist genutzten Diagnosetools. Das Ziel dieser Studie war die Identifikation von sogenannten „antibody mimics“ aus einer Phagenbibliothek gegen das Calibrachoa mottle virus (CbMV), Cucumber mosaic virus (CMV), Plum pox virus (PPV), Potato virus Y (PVY), Tobacco mosaic virus (TMV) und Tomato spotted wilt virus (TSWV). Im Bestfall sollen diese „antibody mimics“ die Vorteile von Antikörpern in einem ELISA besitzen und mögliche Nachteile, wie zum Beispiel die Abhängigkeit der begrenzten Ressourcen, da die benötigten Antikörper ständig nachproduziert und validiert werden müssen, vermieden werden. Dies kann durch die Produktion und Lagerung der „antibody mimics“ in Bakterienzellen erreicht werden. In einem Screeningverfahren, dem sogenannten „Biopanning“, werden Phagen selektiert, die fest an das Zielmolekül binden. In dieser Arbeit wurden diese Biopannings mit den kommerziell erhältlichen Phagenbibliotheken Ph.D.™- 12, Ph.D.™-C7C und den scFv-Bibliotheken Tomlinson I/J ausgeführt. Zusätzlich wurde eine gemischte scFv-Bibliothek aus HAL9/HAL10 verwendet. Neben der bekannten Präsentation der Zielmoleküle in Form von exprimierten Hüllproteinen oder gereinigten Virionen auf Plastikoberflächen wurde außerdem eine Methode getestet, in der die gesamte Bibliothek gegen gesunden Pflanzensaft präinkubiert und danach gegen infizierten Pflanzensaft gescreent wurde. Durch diese Arbeitsschritte wurden eine signifikante Reduzierung der unspezifisch reagierenden Phagen und ein einfacherer und schnellerer Screeningprozess erwartet. Es konnten viele Phagen identifiziert werden, die eine positive Reaktion mit exprimierten Hüllproteinen oder infiziertem Pflanzensaft aufwiesen, die meisten Phagen zeigten jedoch zusätzlich eine Interaktion mit gesundem Pflanzenmaterial. Nur fünf scFvs, die mit der neuen Methode gegen das CbMV gescreent wurden, zeigten eine signifikante Interaktion mit infizierten Nicotiana benthamiana in ELISAs und führten zu starken Signalen ohne eine unspezifische Reaktion mit dem Pflanzenmaterial. Die Ergebnisse weisen darauf hin, dass auf Phagen basierende „antibody mimics“ ein wertvolles ergänzendes Werkzeug für die Verbesserung von Pflanzenvirusnachweisen in ELISAs sein können. Schlagworte: antibody mimic, Pflanzenvirus, Biopanning, ELISA, scFv, CbMV ABSTRACT IV ABSTRACT Despite the continuous development of newer and apparently more advanced methods and techniques for detecting and identifying of plant viruses, only a few of these new methods are used for routine testing in laboratories. Due to the unique characteristics, for example an industry standard format, a robust functionality and high levels of repeatability and reproducibility, the enzyme-linked immunosorbent assay (ELISA) and the real-time polymerase chain reaction (qPCR) are two of the most common used diagnostic tools. The aim of this study was the identification of antibody mimics directed towards the Calibrachoa mottle virus (CbMV), Cucumber mosaic virus (CMV), Plum pox virus (PPV), Potato virus Y (PVY), Tobacco mosaic virus (TMV) and Tomato spotted wilt virus (TSWV) derived from a phage library. These antibody mimics should combine the benefits of antibodies in ELISA procedures but do not have some of the disadvantages, for example the costly dependence on finite resources as the required antibodies for the test procedure have to be produced and validated consistently. To overcome this disadvantage, antibody mimics can be easily produced and stored in bacteria. In a screening procedure called “Biopanning” phages are selected which bind tightly to target molecules. The pannings were carried out with the commercially available phage library Ph.D.™-12, phage library Ph.D.™-C7C, the scFv libraries Tomlinson I/J and a mixed scFv library of HAL9/HAL10. Besides the common offering of expressed virus coat proteins by bacteria or complete virions in Biopanning procedures, we also tested a method in which we preincubated a complete phage library against healthy plant sap and used virus infected plant sap for the following Biopanning. Due to these working steps, we expected a significant loss of cross binding phages and an easier screening process. As a result, many phages of different libraries demonstrated a positive reaction with expressed coat proteins or infected plant sap, but we also noticed interactions with healthy plant material. Only five scFvs, screened with the new method against CbMV, showed a significant interaction with infected Nicotiana benthamiana in ELISAs, revealing high readings within two hours without cross reactions to the plant material. The results indicate that antibody mimics based on phages can be a valuable tool to support and improve ELISAs for detection of plant viruses. Keywords: antibody mimic, plant virus, Biopanning, ELISA, scFv, CbMV INHALTSVERZEICHNIS V INHALTSVERZEICHNIS KURZFASSUNG ................................................................................................................... III ABSTRACT ............................................................................................................................ IV INHALTSVERZEICHNIS ..................................................................................................... V ABKÜRZUNGSVERZEICHNIS ........................................................................................... X Aminosäuren ..................................................................................................................... XIV Virusabkürzungen .............................................................................................................. XV ABBILDUNGSVERZEICHNIS ........................................................................................ XVI TABELLENVERZEICHNIS .............................................................................................. XX 1 Einleitung ................................................................................................................ 24 1.1 Ziele dieser Arbeit .................................................................................................... 24 1.2 Einleitung zu Pflanzenviren ..................................................................................... 25 1.3 Eine kurze Vorstellung der in dieser Arbeit verwendeten Pflanzenviren ................ 28 1.3.1 Calibrachoa mottle virus als Vertreter des Genus Carmovirus ........................... 29 1.3.2 Cucumber mosaic virus als Vertreter des Genus Cucumovirus ........................... 31 1.3.3 Plum pox virus und Potato virus Y als Vertreter des Genus Potyvirus................ 33 1.3.4 Tobacco mosaic virus als Vertreter des Genus Tobamovirus .............................. 37 1.3.5 Tomato spotted wilt virus als Vertreter des Genus Tospovirus ............................ 39 1.4 Bakteriophagen als „antibody mimics“ .................................................................... 41 1.4.1 Eine kurze zeitliche Einordnung der Entdeckung und Geschichte von Bakteriophagen ..................................................................................................... 42 1.4.2 Biologie filamentöser Ff-Bakteriophagen ............................................................ 44 1.4.3 Biopanning mit Phagenbanken ............................................................................. 49 1.5 Der Nachweis von Viren durch „antibody mimics“ aus Phagenbibliotheken ......... 52 2 Material und Methoden ......................................................................................... 54 2.1 Materialien ................................................................................................................ 54 2.1.1 Geräte und Verbrauchsmaterialien ....................................................................... 54 2.1.2 Chemikalien und Reagenzien ............................................................................... 56 INHALTSVERZEICHNIS VI 2.1.3 Verwendete Pflanzenviren .................................................................................... 58 2.1.4 Verwendete Pflanzen ............................................................................................ 59 2.1.5 Lösungen und Puffer ............................................................................................ 59 2.1.6 Verwendete Antibiotika........................................................................................ 62 2.1.7 Escherichia coli Bakterienstämme ....................................................................... 62 2.1.8 Rhizobium radiobacter Bakterienstamm .............................................................. 62
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