Presentation of recombinant proteins in modified vaccinia virus Ankara extracellular enveloped virions [Elektronische Ressource] / vorgelegt von Andrea Meiser

Meiser, Nell Andrea

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2002
• Nombre de lectures: 75
• Poids de l'ouvrage: 13 Mo

Extrait

Presentation of Recombinant Proteins in
Modified Vaccinia Virus Ankara
Extracellular Enveloped Virions
Dissertation
zur Erlangung des Doktorgrades
an der
Fakultät für Biologie
der
LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN
vorgelegt von
Andrea Meiser
Juli 20021. Gutachter: Prof. Dr. L. Gürtler
2. Gutachterin: Prof. Dr. R. Brack-Werner
Sondergutachterin: Dr. J. Krijnse-Locker
Tag der mündlichen Prüfung: 21.2.2003
21. SUMMARY ............................................................................................................6
2. INTRODUCTION....................................................................................................7
2.1 Historical background of vaccination .................................................................7
2.2 Taxonomic classification of poxviruses..............................................................8
2.3 Modified vaccinia virus Ankara (MVA) ...............................................................9
2.4 Genome of vaccinia viruses ............................................................................10
2.5 Life cycle of vaccinia virus...............................................................................12
2.5.1 Adsorption and penetration ......................................................................12
2.5.2 Synthesis of VV-proteins and DNA replication ..........................................14
2.5.3 Assembly.................................................................................................15
2.5.4 Release ...................................................................................................17
2.6 Infectious forms of vaccinia virus.....................................................................18
2.6.1 EEV specific proteins20
2.6.2 Properties of the CEV/EEV envelope .......................................................21
2.7 Modern vaccines ............................................................................................21
2.8 Aim of the work...............................................................................................22
3. MATERIALS ........................................................................................................24
3.1 Abbreviations..................................................................................................24
3.2 Buffers and solutions ......................................................................................25
3.3 Media .............................................................................................................27
3.4 Antibodies ......................................................................................................28
3.5 Cells...............................................................................................................28
3.6 Viruses ...........................................................................................................28
3.7 Oligonucleotides .............................................................................................28
4. METHODS29
4.1 Bacterial techniques .......................................................................................29
4.1.1 Transformation of bacteria with plasmid DNA ...........................................29
4.1.1.1 Preparation of electrocompetent cells................................................29
4.1.1.2 Electrotransformation ........................................................................29
4.1.1.3 Heat shock transformation.................................................................29
4.1.2 Plasmid DNA isolation from E.coli ............................................................30
4.1.2.1 Qiagen maxi preparation ...................................................................30
4.1.2.2 Qiagen mini preparation ....................................................................30
4.2 DNA techniques..............................................................................................31
4.2.1 In vitro modification and recombination of DNA ........................................31
4.2.1.1 Preparation of vectors and inserts by digestion with restriction
enzymes.......................................................................................................31
4.2.1.2 Enzymatic in vitro amplification of DNA by Taq-polymerase ...............32
4.2.1.3 Dephosphorylation of 5‘ ends of DNA fragments with alkaline
phosphatase.................................................................................................32
4.2.1.4 Creation of blunt ends by T4 DNA-polymerase or Klenow enzyme.....32
34.2.1.5 Ligation of DNA fragments by T4 DNA-ligase ....................................32
4.2.2 Purification of nucleic acids ......................................................................33
4.2.2.1 Removal of proteins by phenol/chlorophorm extraction ......................33
4.2.2.2 Concentration of nucleic acids by ethanol precipitation33
4.2.2.3 Photometric estimation of purity and concentration of nucleic acids....33
4.2.2.4 Isolation of DNA fragments from agarose gels (silica adsorption) .......34
4.2.2.5 Purification of PCR products..............................................................34
4.2.3 Analysis of plasmid DNA..........................................................................34
4.2.3.1 Restriction analysis ...........................................................................34
4.2.3.2 Analysis by horizontal agarose gel-electrophoresis............................35
4. 3 Analysis of proteins........................................................................................35
4.3.1 Discontinous denaturating PAA-gel electrophoresis (SDS-PAGE).............35
4.3.2 Identification of proteins by immunoblotting (Western blotting)..................36
4.3.3 Stripping of the Western Blot....................................................................37
3.3.4 Quantification of proteins37
4.4 Cell culture techniques....................................................................................38
4.4.1 Host cells for in vitro cultivation of MVA ....................................................38
4.4.1.1 Preparation of chicken embryo fibroblasts (CEF) ...............................38
4.4.1.2 Cell culture techniques for BHK21 and RK13 cells.............................39
4.4.2 Amplification of Vaccinia Virus..................................................................39
4.4.3 Determination of infectivity .......................................................................40
4.4.3.1 Titration of MVA (TCID )...................................................................4050
4.4.3.2 Titration of MVA by immunostaining...................................................40
4.4.3.3 Titration of WR and IHD-J by cristal violet staining.............................41
4.4.4 Plasmid-DNA transfection of infected adherent cells.................................41
4.4.4.1 Stable transfection for generation of recombinant MVA......................41
4.4.4.2 Transient transfection........................................................................41
4.5 Generation and characterization of recombinant MVA .....................................42
4.5.1 Generation of rMVA by homologous recombination ..................................42
4.5.1.1 Growth selection of recombinant MVA on RK13 cell monolayers........42
4.5.1.2 Deletion of the marker gene K1L .......................................................43
4.5.2 Analysis of recombinant MVA-DNA ..........................................................44
4.5.2.1 Isolation of MVA-DNA .......................................................................44
4.5.2.2 PCR analysis of recombinant MVA-DNA............................................44
4.5.3 Detection of recombinant protein in MVA infected cells.............................45
4.5.3.1 Protein detection by Western blot analysis.........................................45
4.5.3.2 Protein detection by immunostaining .................................................45
4.5.4 Multiple step growth curve........................................................................46
4.5.5 Virus purification on CsCl gradients46
4.5.5.1 Separation and concentration of virus fractions..................................46
4.5.5.2 Sucrose cushion................................................................................47
4.5.5.3 Cesium chloride gradient (Payne and Norrby, 1976) ..........................47
4.6 Electron microscopic analysis .........................................................................47
4.6.1 Immunolabeling and negative staining of viral particles.............................48
4.6.2 Electron microscopic analysis of infected cells..........................................48
4.6.2.1 Infection and drug treatment..............................................................48
4.6.2.2 Fixation .............................................................................................49
4.6.2.3 Epon embedding...............................................................................49
4.6.2.4 Sample preparation...........................................................................49
4.7 Immunofluorescence ......................................................................................50
5. RESULTS .....................................................