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Recombinant expression, purification and characterisation of the surface immunoreceptor CD47 (integrin associated protein)
Kuchyňová, Sarah ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
The human immunoreceptor CD47, also designated <Integrin Associated Protein= is a novel therapeutic target for cancer treatment. When bound to one of its ligands, a protein found on the surface of immune cells, SIRP, the phagocytosis of the CD47 expressing cell is inhibited. This interaction is used by malignant cells to escape detection by the immune system, thus making CD47 a promising candidate molecule for cancer immunotherapy. The literary overview focuses on the characterisation of CD47 in terms of its structure, localization, and function. Additionally, this sections thoroughly delves into the aforementioned interaction with SIRP as well as existing mechanisms for the targeting of the CD47- SIRP axis. The following experimental section centers around the preparation of the plazmid pTT28_hCD47. The process by which the gene for the extracellular portion of human CD47 was inserted into the linearized pTT28 vector is noted and its transfection into a mammalian expression system to produce the protein. Moreover, the section divulges into the purification of the recombinant protein by affinity purification using Ni-NTA agarose as well as gel permeation chromatography, resulting in the isolation of CD47 proteins differing in glycosylation. Their functionality was validated via SPR, in a study...
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Knowledge and Opinions of Students on Genetically Modified Organisms
Semencová, Barbora ; Hlaváčová, Lucie (advisor) ; Vojíř, Karel (referee)
This diploma thesis is focused on topic of genetically modified organisms and their use in the practical sectors of human life. Theoretical part of the thesis defines general terms GMO, plasmid, genetic engineering, biotechnology. It also records historical milestones relating to the problematic, deals with individual techniques of genetic engineering and briefly states legislative procedures in context of dealing with GMO. It gives examples of transgenic organisms and summarizes advantages and disadvantages of their use.Practical part of the thesis contains educational program called "Genetically modified organisms", which was conceived by the author and includes a draft of a lesson inclusive of teaching materials - powerpoint presentations, worksheets, interactive worksheets, auxiliary text for teacher and written preparation. Research part deals with high school students change of view about using GMOs after completing the educational program. Due to analysis was proven that most of the attitudes and knowledge about GMO was changed after completing the educational program (for example in issues of willingness to consume GM food and animal products, perception of advantages and disadvantages etc.) Data was still unchanged in questions which cannot be affected by the program (control of food packaging or...
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Preparation of lentiviral expression vector with reporter gene
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Besides recombinant protein expression in prokaryotic cell lines (E. coli), systems, that could quickly, reliably and stably produce recombinant proteins in human cell lines, come to the fore. These cell lines assure proper tertiary structure and post-translational modification of the desired products. One of the ways to achieve production of recombinant proteins in human cell lines is the use of lentiviral vectors. This thesis describes the preparation of the lentiviral vector (plasmid) Daedalus, which contains a construct for recombinant expression of secreted alkaline phosphatase. For the preparation of the desired plasmid methods based on insertion of the secreted alkaline phosphatase gene using the restriction endonucleases and methods based on amplification by polymerase chain reaction (restriction-free cloning, transfer polymerase chain reaction and Gibson assembly) were used.
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