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Structural analysis of the interaction interface between an antibody and a protein epitope
Moravec, Jan ; Krejčíř, Radovan (referee) ; Müller,, Petr (advisor)
The diploma thesis deals with the study of the mouse monoclonal antibody EEV1-2.1, which was created in the RECAMO laboratories by immunization of mice with the Hsp90 protein and subsequent fusion of splenocytes with a mouse myeloma line. The theoretical part of this work focuses mainly on the study of antibodies, especially their structure, genetics and the description of monoclonal antibodies. In this part of the work, you can also find a cross-section of modern biotechnological trends that are currently used for the production of antibodies. CHO cells and tetracycline inducible systems are also described. The aim of the thesis was to characterize the mentioned antibody. Using bioinformatics methods and prediction software to predict and describe its structure, especially then to analyze hypervariable CDR regions. Another goal was to clone the light and heavy chains of the antibody using the Gateway method. The final step was the creation of a tetracycline inducible system for efficient production of the antibody itself. The experimental part of the work initially deals with the isolation of the RNA sequence of the antibody using commercial kits. Next, the method of amplification of the given sequence using PCR with reverse transcription is described. The following is a description of the Gateway cloning method, which allows convenient insertion of the selected gene into the target vector. The basic bioinformatics methods used to study the structure of antibodies are also explained, as well as the prediction of a more complex 3D structure of the antibody, including possible interactions with the HSP90 protein. This section describes the methods of transfection of ExpiCHO cells and inducible antibody production using the tetracycline inducible system. The thesis led to the successful cloning of the light and heavy chain of the antibody and the subsequent insertion of the expression vector into the production ExpiCHO cells. The expression system based on induction by the addition of doxycycline was then successfully tested by several methods and the results show that the inducible system is not only functional, but can even lead to increased production of monoclonal antibody compared to conventional methods.
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The influence of S159A mutation on the oligomeric state of human NK cell receptor NKR-P1A
Hausleitner, Filip ; Vaněk, Ondřej (advisor) ; Kožíšek, Milan (referee)
Natural killer cells (NK cells) are lymphocytes that possess cytotoxic activity against tumour or virally infected cells independent of preceding antigen sensitisation. To kill such cells, they utilise their activating and inhibitory surface receptors that interact with target cell surface molecules. The immune response carried by NK cells depends on the balance of both activating and inhibitory signals. Human NK cell surface receptor NKR-P1A belongs to the structural family of C-type lectin-like receptors. This receptor interacts with its ligand LLT1, which belongs to the same protein family, with low affinity and high specificity. The NKR-P1A:LLT1 complex formed between NK cell and its target cell inhibits NK cell cytotoxicity, and hence is a part of the regulation of immune response. This thesis studied the effect of S159A mutation on the stoichiometric state of soluble human NKR-P1A ectodomain in solution. Therefore, a mutant form of NKR-P1A G90-S225 S159A ectodomain was successfully produced in stably transfected human embryonic kidney cells 293 (HEK293S GnTI" ). This construct was purified by affinity and size-exclusion chromatography, and analysed by SDS-PAGE and analytical ultracentrifugation. Our results show that the preclusion of N-linked glycosylation in the position 157 promotes the...
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Structural studies of rat NK cell receptor NKR-P1B and its ligand Clrb
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
Natural killer (NK) cells are an intensively studied part of immune system possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. This can be illustrated on the homodimeric rat inhibitory receptor NKR-P1B and its ligand Clrb which play, besides other things, crucial role in the immunological response of NK cells to the infection with rat cytomegalovirus (RCMV), one of the most studied NK cell function model in rat model organism. During RCMV infection the target cell downregulates cell surface expression of Clrb, thus decreasing inhibitory signal transmitted through the NKR-P1B receptor to the NK cell, which would ideally lead to NK cell activation and lysis of the infected cell. However, RCMV carries a gene for "decoy" surface receptor - RCTL that mimics Clrb and thus helps to escape the immunological response of NK cells. Moreover, while this escape strategy was demonstrated in the WAG rat strain, it has been shown that the NKR-P1B homologue from SD rat strain binds only Clrb and does not recognize RCTL. Thus the SD rat strain is less...
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Transgenic technologies based on transposons
Dobiášovská, Ivana ; Kozmik, Zbyněk (advisor) ; Čáp, Michal (referee)
Genenetic engineering is one of the leading technologies in biological research. Transgenesis, one of the most important genetic engineering technologies, enables to study genetic aspects of organismal systems and thus helps us to better understand to the functional characteristics of genomes. Transposons are naturally occuring mobile genetic elements, which can be used to artificially integrate transgenes into host cell genomes. Catalysis of this essential step during transgenesis makes from transposons an useful genetic tool.The aim of this work is to present eukaryotic DNA transposons that transpose in a cut-and-paste-fashion, together with particular mechanisms affecting their function, that can be used as gene delivery system.
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Generation of porcine induced pluripotent stem cells - a model of Huntington disease.
Svobodová, Eliška ; Motlík, Jan (advisor) ; Fulka, Josef (referee)
Stable porcine ES cell lines have not been succesfully established yet. Ability to selfrenew or to differentiate has been limited in different porcine ES-like cell lines so far. PiPSCs represent an alternative to pESCs. PiPSCs can be generated by reprogramming of somatic cells by introduction of several transcription factors on viral vectors and were established by several groups. However, the majority of piPS cell lines depend on transgene expression because of incomplete reprogramming and weak activation of endogenous pluripotency genes. Transgene expression can infuence differentiation potential of piPSCs. Therefore, we have used integrative and reexcisable PiggyBac transposons to generate viral free piPSCs. At the same time, small molecules (low-molecular inhibitors) with potential to increase reprogramming efficiency and to activate endogenous pluripotency genes were used in the reprogramming media. This strategy has a potential for generation of naive piPSCs. Successful excision of transgenes would generate transgene-free piPSCs with uncompromised differentiation potential. Pig (Sus Scrofa) is at the same time an important animal model in preclinical stage research of the diseases. Somatic cells used for generation of piPSCs were isolated from pigs carrying mutated huntingtin. Integration of the...
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