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The Marek's Disease Virus (MDV)

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The Marek's Disease Virus (MDV) Aus dem Institut für Virologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin The Marek’s disease virus (MDV) chemokine vIL8 binds the cCXCR5 receptor and is a functional orthologue of cCXCL13 Inaugural-Dissertation zur Erlangung des Grades eines Doktors der Veterinärmedizin an der Freien Universität Berlin vorgelegt von Ibrahim Mahmoud Alzuheir Tierarzt aus Kuwait Berlin 2016 Journal-Nr.: 3918 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Prof. Dr. Benedikt B. Kaufer Zweiter Gutachter: Univ.-Prof. Dr. Susanne Hartmann Dritter Gutachter: Priv.-Doz. Dr. med. vet. Sonja Härtle Deskriptoren (nach CAB-Thesaurus): Poultry, Marek's disease, avian herpesviruses, genetics, chemokines, in vitro, relationships, proteins, receptors, binding sites, bacterial artificial chromosomes, recombination, virulence, mutants Tag der Promotion: 05.08.2016 Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über <http://dnb.ddb.de> abrufbar. ISBN: 978-3-86387-741-5 Zugl.: Berlin, Freie Univ., Diss., 2016 Dissertation, Freie Universität Berlin D 188 Dieses Werk ist urheberrechtlich geschützt. Alle Rechte, auch die der Übersetzung, des Nachdruckes und der Vervielfältigung des Buches, oder Teilen daraus, vorbehalten. Kein Teil des Werkes darf ohne schriftliche Genehmigung des Verlages in irgendeiner Form reproduziert oder unter Verwendung elektronischer Systeme verarbeitet, vervielfältigt oder verbreitet werden. Die Wiedergabe von Gebrauchsnamen, Warenbezeichnungen, usw. in diesem Werk berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, dass solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürfen. This document is protected by copyright law. No part of this document may be reproduced in any form by any means without prior written authorization of the publisher. Alle Rechte vorbehalten | all rights reserved © Mensch und Buch Verlag 2016 Choriner Str. 85 - 10119 Berlin verlag@menschundbuch.de – www.menschundbuch.de Dedicated To my late father (Abu Nidal) who dreamt of seeing his son a doctor. Table of contents Table of contents .................................................................................................................................. V List of figures and tables.......................................................................................................................X List of Abbreviations: .......................................................................................................................... XII Summary.............................................................................................................................................. .XV 1. Introduction ............................................................................................................................... 1 1.1. Herpesviruses................................................................................................. 1 1.1.1. Taxonomy and classification of herpesvirus.................................................... 2 1.1.2. Structure and genome of Herpesvirus............................................................. 3 1.1.3. Life cycle of herpesvirus.................................................................................. 5 1.2. Marek’s Disease (MD) .................................................................................... 8 1.2.1. Background..................................................................................................... 8 1.2.2. Clinical signsof MD ......................................................................................... 9 1.3. Marek`s Disease Virus (MDV)....................................................................... 10 1.3.1. MDV genome................................................................................................ 10 1.3.2. MDV pathogenesis........................................................................................ 11 1.3.2.1. Infection and early cytolytic phase.............................................................. 11 1.3.2.2. Latent phase .............................................................................................. 12 1.3.2.3. Late cytolytic and immunosuppressive phase............................................. 13 1.3.2.4. Transformation ........................................................................................... 13 1.3.3. Role of viral CXC (vIL-8) in MDV pathogenesis and tumorigenesis............... 14 1.4. Chemokines and chemokine receptors. ........................................................ 15 1.4.1. Chemokine: classification and nomenclature ................................................ 16 VI Table of contents 1.4.1.1. Chicken`s CXC chemokines....................................................................... 17 1.4.1.2. Chicken CXCL8 (cIL8)................................................................................ 19 1.4.1.3. Chicken CXCL13........................................................................................ 20 1.4.1.4. MDV CXC chemokine (vIL-8) ..................................................................... 20 1.4.2. Chemokine receptors.................................................................................... 21 1.5. Project introduction. ...................................................................................... 24 1.5.1. Evolutionary and functional characterization of vIL-8. ................................... 24 1.5.2. Role of vIL-8 in MDV replication and tumor formation ................................... 25 2. Materials and Methods ........................................................................................................... 26 2.1. Materials ....................................................................................................... 26 2.1.1. Established cell lines, bacterial strains and insect cell................................... 26 2.1.2. Chemicals and reagents ............................................................................... 26 2.1.3. Equipments and consumables ...................................................................... 28 2.1.4. Media and buffers. ........................................................................................ 30 2.1.5. Kits................................................................................................................ 32 2.1.6. Enzymes....................................................................................................... 32 2.1.7. Plasmids, bacmids and BACs. ...................................................................... 32 2.1.8. Primers and probes....................................................................................... 35 2.1.9. Antibodies..................................................................................................... 38 2.1.10. Software ....................................................................................................... 38 2.2. Methods........................................................................................................ 39 2.2.1. Standard Molecular biological methods......................................................... 39 2.2.1.1. Bioinformatics analysis............................................................................... 39 2.2.1.2. Phylogenic trees and sequence analyses................................................... 39 2.2.1.3. Polymerase Chain Reaction (PCR) ............................................................ 41 Table of contents VII 2.2.1.4. DNA isolation and purification..................................................................... 43 2.2.1.5. Agarose gel electrophoresis ....................................................................... 43 2.2.1.6. Restriction endonuclease. .......................................................................... 43 2.2.1.7. DNA gel extraction ..................................................................................... 44 2.2.1.8. Determination of DNA-concentration by spectrophotometry ....................... 44 2.2.1.9. Dephosphorylation ..................................................................................... 44 2.2.1.10.DNA ligation ............................................................................................... 44 2.2.1.11.Colony PCR ............................................................................................... 44 2.2.1.12.DNA sequencing ........................................................................................ 45 2.2.2. Microbiological Methods................................................................................ 46 2.2.2.1. Bacterial culture.......................................................................................... 46 2.2.2.2. Preparation of chemically competent E. coli (TOP10 and DH10B) ............. 46 2.2.2.3. Chemical transformation of chemically competent E. coli ........................... 46 2.2.2.4. Preparation of electrocompetent E.coil. ...................................................... 47 2.2.2.5. Small-scale plasmid DNA isolation from bacteria (Mini-prep)...................... 47 2.2.2.6. Medium-Scale plasmid DNA isolation from bacteria
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