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Effect of polyhistidine modification of viral particles on their intracellular localization and gene delivery to the nucleus
Číhařová, Barbora ; Španielová, Hana (advisor) ; Grantz Šašková, Klára (referee)
Viral vectors derived from mouse polyomavirus are a convenient tool for studying the targeted delivery of therapeutical agents into the cells and cellular organelles. Vectors derived from mouse polyomavirus face difficulties similar to other nanoparticles, as they often end up trapped inside an endosome where they are subsequently degraded. This diploma explored the potential of vector modifications, which have the potential to make the transport to the nucleus or cytosol more effective. This work had particularly focused on increasing the transduction efficiency by modifying particle's internally localized VP3 capsid protein with covalently bound membrane-penetrating peptides. Primary covalent genetic modification to the VP3 protein was the polyhistidine peptide KH27K. Its potential of improving the transduction effectivity was compared with two other peptide modifications - LAH4 and R8. The results of the transduction test showed that covalently bound R8 peptide had many-fold improved the transport to the nucleus when compared to the unmodified particles. The modification with LAH4 peptide had been regarded more effective only when was associated with the particles non-covalently. In such scenario the transduction efficiency rose 40-times when compared with unmodified particles. Polyhistidine...
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Experimental system for production of IL-15 on viral carriers
Musil, Dominik ; Španielová, Hana (advisor) ; Šmahel, Michal (referee)
Interleukin 15 has great application potential such as in the biological treatment of cancer. It is involved in a variety of immunological processes, the most important of these involve influencing and induction of NK cells and T-lymphocytes proliferation. However, its therapeutic usages are limited by a low stability and short half-life. For this reason, there are various approaches of stabilization and expansion of its biological activity being explored. In this work, we analysed and developed a new approach, which uses viral nanostructures derived from major capsid VP1 protein of mouse polyomavirus as a carrier of IL-15. Moreover, VP1 proteins can be relatively easily modified and they are also capable to penetrate into the tumour cells. There were prepared two variants of IL-15 together with control nanostructures in the baculovirus expression system, one was composed of IL-15 and the other of the IL-15 fusion protein and truncated variant of VP1. Protein constructs were characterized by electron microscopy and biochemical methods. The total protein yield of VP1ΔC-IL15-HIS fusion variant was higher (up to 53 mg/L of complete medium) than IL-15 alone (8,5 mg/L). However, testing of the biological activity of the prepared proteins in vitro did not show any induction of proliferation on Jurkat...
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Development of a technique for gene transfer into T-lymphocytes using polyomavirus structures and the LAH4 peptide
Schreiberová, Lucie ; Španielová, Hana (advisor) ; Vopálenský, Václav (referee)
Efficient delivery of genetic material to T-lymphocytes is key in gene therapy using T-lymphocytes with chimeric antigen receptors. Current procedures require the use of potentially dangerous viral vectors or large amount of input material. The diploma thesis therefore focuses on exploring new approaches for gene transfer into T-lymphocytes: use of safe virus-like particles (VLPs) derived from mouse polyomavirus in combination with the amphipathic cationic peptide LAH4. LAH4 has the potential to increase the efficiency of DNA and viral vector transport into cells. The system which combines VLPs and the LAH4 peptide was optimized for the delivery of reporter gene (encoding GFP and luciferase) to the model T-cell line Jurkat. It has been found that Jurkat cells cannot be efficiently transduced by DNA packed into VLPs. When cells were transfected only with DNA and LAH4, consistent results were not obtained, and the transfection efficiency ranged from 0.5 to 19%. The diploma thesis also analysed the effect of phosphorylation of viral structures on gene transfer. The impact of treatment of virus particles by alkaline phosphatase on the infectivity of the virus was studied and it was necessary to analyse the effect of the reaction components. Sublytic concentration of Triton-X100 in the reaction buffer...
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Preparation of Monoclonal Antibodies Against VP2 Protein of Human Polyomaviruses
Vochyánová, Klára ; Drda Morávková, Alena (advisor) ; Růžičková, Šárka (referee)
Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...
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Construction of mouse polyomavirus chimeric VLP bearing melanoma epitopes
Kojzarová, Martina ; Drda Morávková, Alena (advisor) ; Tachezy, Ruth (referee)
Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...
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The use of plants for the expression of Human papillomavirus vaccine
Dlabalová, Lucie ; Moravec, Tomáš (advisor) ; Fischer, Lukáš (referee)
Papillomaviruses are causing various diseases from skin warts to the lesions leading to malignant tumours and are widespread among people. For this reason, the current research is trying to develop methods for the production of inexpensive and effective vaccines against both Papillomaviruses and against all other infectious diseases. Currently animal and microbial expression systems are most frequently used for the production of biopharmaceuticals which have several drawbacks and their capacity is limited. This opens up the doors for plants - potentially very efficient producers of biopharmaceuticals. Currently there is rapid development towards the optimization and improvement of the results of plant expression systems and establishing the best and safest methods of their use. This paper summarizes and compares the advantages and disadvantages of different methods of plant transformation, leading either to stable production of the protein of interest in transgenic plants or to transient expression of recombinant virus infecting non-transgenic plants. Furthermore it analyzes the most appropriate plant species, which provide high yields combined with a transformation method and ease of cultivation, describes few basic ways of optimizing expression levels and outlines the future of plant expression systems.
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