Studies on reverse genetic systems for avian influenza virus and the Borna disease virus [Elektronische Ressource] / vorgelegt von Wenjun Ma

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2003
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem Institut für Medizinische Virologie
der Justus-Liebig-Universität Gießen
Betreuer: HDoz Dr. Stephan Pleschka









Studies on reverse genetic systems
for avian influenza virus and the Borna disease virus



INAUGURAL-DISSERTATION
zur
Erlangung des Doktorgrades
der Naturwissenschaftlichen Fachbereiche
der Justus-Liebig-Universität Gießen



vorgelegt von
Wenjun Ma
geb. 03. 10.1972 in Heilongjiang China



Gießen, 2003













Mit Genehmigung des Fachbereichs Biologie
der Justus-Liebig-Universität Gießen




Dekan: Prof. Dr. Jürgen Mayer


1. Gutachter: HDoz Dr. Stephan Pleschka
Institut für Medizinische Virologie
Justus-Liebig-Universität Gießen

2. Gutachter: Prof. Dr. Gabriele Klug
Institut für Mikrobiologie und Molekularbiologie
Justus-Liebig-Universität Gießen









I
Contents
Zusammenfassung V
Summary XIII
Introduction 1
1 Avian influenza and influenza A virus 1
1.1 Avian influenza ......................................................................................................1
1.1.1 History ...........1
1.1.2 Current situation.............................1
1.1.3 Clinical symptoms ..........................................................................................3
1.2 Influenza A virus.....................................3
1.2.1 Morphology and genome structure of influenza A virus3
1.2.2 Propagation and genome replication of influenza A virus .............................5
1.2.3 The relation between human flu epidemics and AIV.....8
2 Borna disease 10
2.1 Host range and clinical symptoms ........................................................................11
2.2 BDV......................................................11

3 The progress of reverse genetics systems for negative-strand RNA viruses 15
3.1 Influenza virus.......................................................................................................15
3.2 Nonsegmneted negative-strand RNA-viruses (NNS viruses) ...............................17
Materials and methods 23
1 Materials 23
1.1 Chemicals and reagents.........................................................................................23
1.2 Enzymes and enzyme inhibitor.............24
1.3 Nucleotides and reaction buffer ............................................................................24
1.4 Kits........................................................24
1.5 Materials for cell culture.......................................................................................25
1.6 E. coli strains and cell lines...................25
1.7 Plasmids ................................................25
1.8 Antisera and monoclonal antibodies.....................................................................26
1.9 DNA oligonucleotides ..........................................................26
II

1.10 Other materials....................................................................................................28
2 Methods 28
2.1 DNA cloning and subcloning................................................................................28
2.1.1 Preparation of competent cells for eletroporation........28
2.1.2 Electroporation.............................................................................................29
2.1.3 Preparation of plasmid DNA........................................30
2.1.4 Restriction endonuclease digestion..............................31
2.1.5 Filling recessed 3' terimi by Klenow fragment of DNA polymerase I.........31
2.1.6 Dephosphorylation .......................................................................................31
2.1.7 Phenolization and precipitation DNA..........................31
2.1.8 Agarose gel electrophoresis..........................................................................32
2.1.9 Preparation of DNA fragments.....32
2.1.10 Ligation.......................................32
2.2 Plasmids construction...........................................................32
2.2.1 Plasmid PCR.................................32
2.2.2 RT-PCR........................................................................33
2.2.3 pPol1HHRCAT2.1#1, #2 and #3, pPol1HHRCAT2.2#1, #2 and #3...........34
2.2.4 pcDNA3.1Ribo1p, pcDNA3.1Ribo1s-p, pcDNA3.1Ribo2 and pcDNA3.1-
Ribo3............................................34
2.2.5 pPCRII-TOPO-RPA.....................................................34
2.2.6 pPOLIHHR- T7.............................................................35
2.2.7 pBD...............................................35
2.2.8 pBD-PB1, -PB2, -PA....................35
2.2.9 pBD-NP........................................................................35
2.2.10 pBD-HA, -NS.............................................................35
2.2.11 pBD-NA, -M...............................36
2.3 Ribozyme assay.....................................................................36
2.3.1 Plasmid linerization......................................................36
2.3.2 T7 - transcription (in vitro)...........36
2.3.3 Ribozyme reaction........................................................36
2.3.4 Running denaturing acrylamide gel..............................37
2.3.5 Silver staining ...............................................................37
2.4 Indirect Immunofluoresces Assay (IFA) and in situ immunhistochemical
BDV-detection.......................................................................38
2.4.1 IFA................................................38
2.4.2 In situ immunhistochemical BDV-detection39
2.5 Establishment of Vero cell line infected by BDV H1766.....................................39
2.6 DNA-transfection of eucaryotic cell cultures .......................................................40
2.6.1 Transfection I; Normal Lipofectamine Reagent...........40
2.6.2 Transfection II; Lipofectamine 2000............................40
2.7 Generation, amplification and purification of wild and reassortant avian
influenza virus.......................................................................................................41
III
2.8 Chloramphenicol Acetyltransferase (CAT)-Assay...............................................42
2.8.1 Preparation of cell extracts ...........................................42
2.8.2 Determination of protein amount.................................42
2.8.3 CAT-Assay...................................42
2.9 Plaque-Assay.........................................43
2.9.1 Plaque-Assay................................43
2.9.2 Analysis of Plaque-Assay.............44
2.10 Haemagglutination (HA) assay...........................................44
2.10.1 Preparation of red blood cells (RBCs) from chicken blood .......................44
2.10.2 HA-Assay...................................................................44
2.10.3 Determination of HA-Units........45
2.11 Luciferase activity assay.....................................................................................45
2.11.1 Preparation of passive lysis buffer.............................45
2.11.2 Active lysis of cultured cells ......................................................................45
2.12 Western Blotting (Semi-dry)...............45
2.12.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE).............................45
2.12.2 Transfer membrane in "Semi-dry" electroblotter .......................................47
2.12.3 Antibody-incubation...................................................47
2.12.4 ECL-reaction..............................................................48
2.13 RNase protection assay (RPA)............................................48
2.13.1 Synthesis of the biotin labeled probe..........................48
2.13.2 Gel purification of probe............................................49
2.13.3 RNA preparation and purification..............................49
2.13.4 Hybridization and RNase digestion of probe and sample RNA.................50
2.13.5 Separation and detect