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Regīna Renhofa (Read 16052 times)
Regīna Renhofa
15.05.2007 at 16:56:13
 
Virus-like particles
as tools for exposition of foreign protein sequences and for delivery of immunomodulators
 
 
Background:
 
The structure of RNA bacteriophages is very simple. They form icosahedral capsids which enclose a single-stranded RNA molecule that contains all the information necessary for the virus to reproduce. Using methods of genetic engineering, recombinant RNA phage capsids were obtained. Such particles can no longer infect bacteria, can not reproduce themselves, but retain their characteristic structure - icosahedral particles formed by 180 coat protein molecules. Among the first such recombinant particles were MS2 (Robert A. Mastico et al, 1993, UK), fr and Qβ (Paul Pumpen et al, 1986 and 1993, Latvia, BMC). Qβ particles were produced as mosaics: like wild type capsids, they contained some copies of prolonged coat protein. This opened possibilities to express foreign protein sequences and expose them on the surface of the particles. Currently we are working with particles produced from coliphages fr, GA, Qβ and phage AP 205 from Acinetobacter (J. Klovins et al, 2002, Latvia, BMC).
 
Our approaches to VLP production:
 
1) VLPs are obtained by expressing native RNA bacteriophage coat protein and a coat protein containing foreign sequence from two different plasmids;
 

 
 
2) VLPs are obtained by expressing native RNA bacteriophage coat protein and a coat protein containing foreign sequence from one plasmid containing both genes;
 
 
 
3) VLPs are obtained by expressing native RNA bacteriophage coat protein and a coat protein containing foreign sequence from one plasmid using opal and amber suppression;
 

 
4) VLPs are obtained by expressing only a coat protein containing N-terminaly or C-terminaly attached foreign sequence and obtaining particles where each monomer contains this foreign sequence;
 

 
5) VLPs are obtained in vitro – by artificially assembling particles from isolated and purified proteins – coat proteins of native RNA bacteriophage and coat proteins containing foreign sequence.
 

 
 
Purification:
 
Individual purification scheme is adapted for each product. Methods used in this process include: fractionation with ammonium sulfate and polyethylene glycol, gel-filtration using Sepharose 2B, 4B and 6B, sucrose density and CsCl density gradient unltracentrifugation, ion-exchange column chromatography, concentration using Amicon ultrafilters and dialysis.
 
 
Studies:
 
Studies on the dissociation-reassociation of VLPs opened up new opportunities of using VLPs as new-generation drugs. Conditions for disassembly and reconstruction of different recombinant capsids have been established and the ability to produce chimeric capsids (consisting of different proteins) in vitro has also been shown. Currently the potential of packaging and its optimization is being studied extensively (Regīna Renhofa).
VLPs can be used to expose certain eucaruotic epitopes, for example, different HBV preS sequences, or they can act as agonists-antagonists for cell receptors. We believe that by recognition of receptors and interaction with them VLPs could be internalized and deliver desired drugs or immunomodulators inside the cell.
 
 
Grants:
 
Regīna Renhofa’s work is being supported by grants 04.1141 “Virus-like recombinant chimeric particles formed by phage Qβ coat protein and HBV surface protein – studies on interaction with hepatocyte cell cultures” and 05.1629 “Virus-like particles formed by RNA bacteriophage coat proteins – reconstruction and packaging in vitro” of Latvian Council of Science.
Renhofas's staff takes part in the Scientific Latvian Staats-programm with project "Artifical virus-like particles as a new transport form for the bioactive substances".
 
 
Our publications:
 
1) Schwarz K., Meijerink E., Speiser D., Tissot A., Cielens I., Renhofa R., Dishlers A., Pumpens P. and Bachmann M. Effiecient homologous prime-boost strategies for T cell vaccination based on virus-like particles. - European Journal of Immunology, 35: 816-821, 2005.
 
2) Shestakova I., Domracheva Ilona, Shkutele S., Ose-Klinklava V. and Renhofa R. Interaction of mosaic virus-like particles carrying HBV preS sequences with cell surface in cultures: Do these epitopes work as specific addresses? – In: Riga meeting on comprehensive cell biology, Riga, June 2-4, 2005.
 
3) Shkutele S., Cielens I., Skrastina D. and Renhofa R. Development of mosaic virus-like particles on base of RNA phage Qβ coat and preS sequences. - Cell Biology International, 29, 111, 35-P3, 2005.
 
4) Strods A., Rumnieks J., Cielens I. and Renhofa R. Development of RNA bacteriophage-based virus-like particles addressed with stromal cell derived factor (SDF-1) sequences for interaction with chemokine receptor CXCR4. - Abstract, submitted to Keystone Symposia Conference, USA, March 2007.
 
 
Our team:
 
Basic team:
 
Regina Renhofa, team leader:
Eduards Urbanovičs:
Dace Priede:



Arnis Strods:
Jānis Rūmnieks:



In collaboration with:
 
Indulis Cielēns:
Ludmila Jackeviča:
Ināra Akopjana:




 
 
« Last Edit: 16.05.2007 at 12:28:24 by a.strods »  
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