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In planta production of TMV (Tobacco mosaic virus) nanoparticles of specific length
Dlabalová, Lucie ; Moravec, Tomáš (advisor) ; Šafářová, Dana (referee)
Tobacco mosaic virus (TMV) is one of the most investigated viruses and its attributes and structure are therefore well-known. In this work, we have chosen TMV as a biotemplate for the adjustable-length particles production in plants. The viral RNA and coat protein of TMV self-assemble into particles under physiological conditions. The particle length depends on the length of packaged RNA. The encapsidation signal that is necessary for preferential viral RNA packaging by coat protein disks is known and characterized since the 1980's. In this work, we have proposed a two-component system based on a Nicotiana bentamiana plants infection with packaging competent defective RNA (dRNA) and a helper virus RNA which provides all the components necessary for dRNA replication and packaging. The encapsidation signal in the helper virus sequence was removed to avoid formation of particles of incorrect length. Some of our helper viruses contained a coat protein with modified region of the particle's inner channel. This modification should allow specific binding of metal atoms within the core of the rod shaped particle. Several variants of dRNA and helper viruses were prepared to identify individual areas important for the replication, encapsidation and nanoparticle stability. We focused on the particle formation...
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Coat protein-RNA interaction in vivo and the biotechnological use of VLPs
Kratochvílová, Kateřina ; Moravec, Tomáš (advisor) ; Hála, Michal (referee)
The Tobacco mosaic virus (TMV) is a simple and frequently used model virus which has been studied already more over than 130 years. Due to the intensive study of this virus the details of its infectious cycle, genomic information and also the structure of the created viral particle as well as the mechanism of its creation are known today. The process of encapsidation (viral particle formation) is sufficiently described in the in vitro conditions. In the in vitro conditions the origin of assembly (OAS) was also described. The OAS was identified in the coding sequence of the gene for the movement protein (MP). The importance of replication centers (replication factories) has also been supposed. The aim of the diploma thesis was to study the specificity of the interaction of RNA and coat protein in the process of the particle assembly taking place directly inside the plants. The experiments were performed to verify the necessity of presence of OAS sequence in process of initiation of viral encapsidation. The effect of the cell compartmentation on this process has also been studied. Based on several viral systems (the Tobacco mosaic virus, the Potato virus X, the Bean yellow dwarf virus and Cowpea mosaic virus) gene constructs were created. These constructs enables to study this idea at the molecular...
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Viral vectors based on tobacco mosaic virus for the expression of recombinant proteins in plants.
Kroupa, Daniel ; Moravec, Tomáš (advisor) ; Plchová, Helena (referee)
(eng): Over the past few decades, plant viruses and vectors derived from them have become not only an integral part of the work in laboratories, but also began to be used commercially for the production of vaccines, drugs, enzymes and other proteins / peptides. One of the most commonly used viruses for the expression of heterologous proteins is tobacco mosaic virus. In this work, I would like to focus on plant viruses and vectors derived therefrom. Compare it with other expression systems used in plants and describe how their benefits could be further used and combined with modern methods of cloning. Key words: viral vector, expression, Tobacco mosaic virus, Cowpea mosaic virus, Golden gate cloning
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In planta production of TMV (Tobacco mosaic virus) nanoparticles of specific length
Dlabalová, Lucie ; Moravec, Tomáš (advisor) ; Šafářová, Dana (referee)
Tobacco mosaic virus (TMV) is one of the most investigated viruses and its attributes and structure are therefore well-known. In this work, we have chosen TMV as a biotemplate for the adjustable-length particles production in plants. The viral RNA and coat protein of TMV self-assemble into particles under physiological conditions. The particle length depends on the length of packaged RNA. The encapsidation signal that is necessary for preferential viral RNA packaging by coat protein disks is known and characterized since the 1980's. In this work, we have proposed a two-component system based on a Nicotiana bentamiana plants infection with packaging competent defective RNA (dRNA) and a helper virus RNA which provides all the components necessary for dRNA replication and packaging. The encapsidation signal in the helper virus sequence was removed to avoid formation of particles of incorrect length. Some of our helper viruses contained a coat protein with modified region of the particle's inner channel. This modification should allow specific binding of metal atoms within the core of the rod shaped particle. Several variants of dRNA and helper viruses were prepared to identify individual areas important for the replication, encapsidation and nanoparticle stability. We focused on the particle formation...
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