A single chain antibody against a viral RNA polymerase (TBSV-BS3-Statice) [Elektronische Ressource] / Kajohn Boonrod

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Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2003
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

A single chain antibody against a viral RNA polymerase (TBSV-BS3-Statice)
Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften
am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz
Kajohn Boonrod
Geb. in Bangkok, Thailand
Neustadt an der Weinstrasse, 2003Tag der mündlichen Prüfung: 25. Juli 2003to
my parents
and
my teachersAcknowledgement
To say “Thanks”, it is too general to express my appreciation to the following persons who
contributed their energy, help, knowledge on this work. However, I would like to thank:
Ulrike Raulfs who supports me and suggested me to keep studying.
Dr. H.-P. Lorenz, the director of the Staatliche Lehr und Forshungsanstalt für Landwirtshaft,
Weinbau und Gartenbau Berufsbildende Schule (SLFA, Neustadt) and Dr. Gabi Krczal, the
director of Centrum Grüne Gene technik (CGG) who offered me the opportunity to work on
this project. Moreover I would also like to thank Dr. Gabi Krczal for her kindness to supervise
me and to give me every kind of supports which brought me to achieve the aim of this project.
Dr. Danuta Galetzka who contributed her knowledge and help on this work.. I truly believe
that without her I could not have finished this work. I also would like to thank her for her
kindness to take care of me during my working in CGG.
Priv.-Doz. Dr. Udo Conrad and Dr. P. D. Nagy who provided their knowledge on phage
display and scFv-mediated RdRp inhibition assays respectively. Moreover I would like to
thank them to allow me to work in their labs.
Michele Frizt for her help in tissue culture, Sasitorn Schotiwutmontri for computerisation and
taking care of transgenics plants. Christian Naumer for his plasmid maps.
Dr. G. Mönke, Dr. J. Pogany, I. Tillak and K.S. Rajendran for their valuable discussion and
help.Table of Contents
Abbreviation........................................................................................................................1
I. Summary........................................................................................................................ 2
II. Introduction....................................................................................................................5
Symptomatology, Host Range and Geographic Distribution of Tombusviruses......... 8
Transmission.................................................................................................................9
Genome structure and molecular biology.....................................................................9
The role of the different proteins encoded by the Tombusvirus genome
in the viral replication cycle......................................................................................... 11
1. The 33K and 92K proteins..................................................................................11
2. The 41 kD protein...............................................................................................11
3. The 22 kD and 19 kD proteins............................................................................12
Viral replication............................................................................................................13
Viral RdRp....................................................................................................................15
III. Materials and methods...................................................................................................18
Molecular cloning.........................................................................................................18
1. Cloning of RdRp fragments.....................................................................................18
1.1 Amplification of the DNA fragments...............................................................18
1.2 Low-melting agarose extraction....................................................................... 19
1.3 Ligation.............................................................................................................20
1.4 Transformation................................................................................................. 20
1.4.1 Competent bacteria................................................................................. 20
1.4.2 Transformation....................................................................................... 21
1.5 Mini-preparation of plasmid DNA................................................................... 22
1.6 Digestion of cloned plasmids............................................................................22
1.7 Purification of plasmid..................................................................................... 22
1.8 Agarose gel electrophoresis..............................................................................23
1.9 Sequencing........................................................................................................23
2. Bacterial expression of RdRp fragments.................................................................24
2.1 Induction...........................................................................................................24
2.2 SDS-PAGE gel................................................................................................. 24
2.3 Soluble and insoluble protein determination.................................................... 25
2.3.1 Shearing with syringe method................................................................ 25 2.3.2 By using BugBuster® reagent................................................................ 25
2.3.3 Osmotic shock........................................................................................ 26
2.4 Analysis of expressed proteins......................................................................... 26
2.4.1 Western blot analysis..............................................................................26
2.4.2 Immunodetection with anti-His antibodies.............................................27
3. Purification of E. coli expressed RdRp fragments.................................................. 27
4. Phage display for scFvs selection............................................................................27
4.1 Tritation of phage library..................................................................................27
4.2 Selection of scFvs.............................................................................................28
4.3 Monoclonal phage ELISA................................................................................ 29
4.4 Soluble expression of scFvs..............................................................................29
4.5 Purification of soluble expressed scFvs............................................................30
5. Antibody-mediated RNA dependent RNA polymerase inhibition assay................30
5.1 In vitro assay.....................................................................................................30
5.1.1 Preparation of RNA templates................................................................30
5.1.2 ScFv-mediated inhibition of RdRp.........................................................32
5.2 In vivo assay......................................................................................................32
5.2.1 In vivo assay by Agroinfiltration method............................................... 33
5.2.1.1 Cloning............................................................................................33
5.2.1.2 Agro-infiltration, intact leaves method...........................................33
5.2.1.2.1 Preparation of Agrobacteria suspension...............................33
5.2.1.2.2 Infiltration of intact leaves....................................................34
5.2.1.2.3 Challenging the infiltrated plants with virus particles.......... 34
5.2.2 In vivo assay by using a virus based vector............................................ 34
5.2.2.1 Cloning the scFvs gene into the infectious clone........................... 34
5.2.2.2 In vitro transcription....................................................................... 35
6. ELISA detection of the binding of scFvs to HCV NS5B RdRp............................. 35
7. Total protein extraction from plant material........................................................... 36
8. Establishment of N. benthamiana transgenic plants expressing scFvs................... 36
9. Challenge inoculation of transgenic plants with viruses......................................... 37
IV. Results.......................................................................................................................... 38
1. Target selection....................................................................................................... 38
2. Cloning of TBSV RdRp fragments......................................................................... 39
3. Expression of the RdRp fragments in E. coli.......................................................... 40 3. Determination of protein expression....................................................................... 43
4. Wes