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Study of the interaction of receptor NKp46 with adhesin Epa1
Houserová, Jana ; Vaněk, Ondřej (advisor) ; Pavlíček, Jiří (referee)
One of the key components of the innate immune system are natural killer (NK) cells. The task of these cells is to induce apoptosis in target cells (e.g., cancer or virally infected cells). The target cells are identified by their interaction with surface receptors of the NK cells. On the surface of the NK cells, there are activating and inhibiting receptors. One of the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands of this receptor have been identified, one of them being the epithelial adhesin Epa1 of yeast Candida glabrata. The invasive candidiasis caused by this yeast is a feared complication for patients with haematological diseases. The use of the NK cells in immunotherapy includes bispecific fusion proteins which can bind to the NK receptor with one part and to tumour antigen with the other part. This work focuses on recombinant preparation of the NKp46 protein. To facilitate a study of the effects of O-glycosylation on the binding of the ligands, a mutation of the glycosylation site NKp46 T225A was prepared. A stably transfected HEK293S GnTI- and HEK293T cells had been prepared and these proteins were then extracellularly secreted. The Epa1 protein had been produced in E. coli bacterial expression system and purified. The binding ability of the Epa1 protein...
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Study of the interaction of receptor NKp46 with adhesin Epa1
Houserová, Jana ; Vaněk, Ondřej (advisor) ; Pavlíček, Jiří (referee)
One of the key components of the innate immune system are natural killer (NK) cells. The task of these cells is to induce apoptosis in target cells (e.g., cancer or virally infected cells). The target cells are identified by their interaction with surface receptors of the NK cells. On the surface of the NK cells, there are activating and inhibiting receptors. One of the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands of this receptor have been identified, one of them being the epithelial adhesin Epa1 of yeast Candida glabrata. The invasive candidiasis caused by this yeast is a feared complication for patients with haematological diseases. The use of the NK cells in immunotherapy includes bispecific fusion proteins which can bind to the NK receptor with one part and to tumour antigen with the other part. This work focuses on recombinant preparation of the NKp46 protein. To facilitate a study of the effects of O-glycosylation on the binding of the ligands, a mutation of the glycosylation site NKp46 T225A was prepared. A stably transfected HEK293S GnTI- and HEK293T cells had been prepared and these proteins were then extracellularly secreted. The Epa1 protein had been produced in E. coli bacterial expression system and purified. The binding ability of the Epa1 protein...
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Recombinant expression of protein NKp46 and its ligand Epa1
Houserová, Jana ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Natural killer cells (NK cells) are cells of innate immunity that play an essential role in the immune response of an organism. In contact with infected, stressed or tumour cells, the NK cells can trigger cytotoxic mechanisms. The initiation of mechanisms depends on the presence of activating or inhibitory ligands on the surface of the cells. On the surface of the NK cells, there are activating and inhibiting receptors that upon binding their respective ligands send a signal to the NK cell. One of the activating mechanisms is the decrease of expression in MHC gp I molecules on the surface of the infected cells. This molecule is a ligand of the inhibiting receptors. One of the activating receptors of NK cells is NKp46. This receptor belongs to the natural cytotoxicity receptor (NCR) family. NKp46 has many ligands, one of them being the adhesin Epa1 of yeast Candida glabrata. This thesis aims at preparing plasmids and producing extracellular domains of the NKp46 receptor and its ligand adhesin Epa1.The interaction of NKp46 and its ligand Epa1 remains to be the subject of future research. Plasmids containing NKp46 and Epa1 genes were successfully prepared and verified by DNA sequencing. The NKp46 protein was produced in eukaryotic expression system of HEK293S GnTI- cell line. A stably transfected...
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Recombinant preparation of proliferating cell nuclear antigen (PCNA)
Herynek, Štěpán ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer (NK) cells are cells of native immunity. Their task is to recognize and destroy tumor cells or, for example, viruses infected cells. NK cells use a number of surface receptors to recognize affected cells. These receptors may be activating, i.e., activate the cytotoxic response of NK cells leading to the induction of apoptosis in the target cell, or inhibitory ones, which, on the other hand, inhibit NK cell response. Whether a cytotoxic response occurs depends on both types of NK receptors. The NKp44 receptor belongs to the family of immunoglobulin receptors and it is one of the activating receptors. Its properties are very unique among other receptors. Other activating receptors of the immunoglobulin receptor family are NKp46 and NKp30. Recently, the interaction between the NKp44 receptor and the proliferating cell nuclear antigen (PCNA) has been described. PCNA is a clamp protein that plays a key role in DNA replication and repair as it anchors replication proteins. This work focuses on the production and optimization of a production of PCNA and on the preparation of expression vectors for the later production of NKp44 and NKp46. Interaction of NKp44 and PCNA will be investigated later. For the same reason, the expression vector for the NKp46 protein is also prepared in this work....
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