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Recombinant expression of the mouse NK cell receptor NKR-P1C
Rudenko, Anastassiya ; Vaněk, Ondřej (advisor) ; Čermáková, Michaela (referee)
NK cells are at the forefront of the body's defense against various threats, such as viruses or cancer cells. NK cells can recognize and destroy harmful cells in several ways that are very different from the defense mechanisms of, for example, T- or B-lymphocytes. NK cells carry a wide range of activating and inhibitory receptors on their surface that distinguish healthy cells from harmful or infected cells. NKR-P1 (natural killer receptor-protein 1) is the first discovered family of NK cell receptors similar to C-type lectins, which includes both activating and inhibitory receptors. NKR-P1C is an activating receptor capable of forming homodimers on the surface of NK cells. The major ligands of the NKR-P1 receptor family are the immunoenvasins of murine cytomegalovirus and other molecules structurally distinct from MHC class I glycoproteins. This work focuses on the recombinant expression of the NKR-P1C receptor protein. Using the pUT7 plasmid, we prepared a stably transfected HEK293T cell line carrying the gene for expression of the extracellular domain of NKR-P1C fused to the Fc fragment of IgG using the PiggiyBac system, and between them, they carry the TEV protease cleavage site. The protein was purified by affinity and gel permeation chromatography, and its purity was subsequently verified by...
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Expression of the recombinant extracellular parts of leukocyte receptors AICL a NKR-P1CBALB
Čonka, Martin ; Novák, Petr (advisor) ; Cebecauer, Marek (referee)
v anglickém jazyce NK cells represent a population of lymphocytes which are able to kill certain tumor cells or virally infected cells. The subject of the diploma thesis is a mouse NK cell receptor mNKR- P1CBALB and a human leukocyte receptor hAICL. The mNKR-P1CBALB belongs to the activating receptors and is able to activate the cytotoxic functions of NK cells. The hAICL receptor is a ligand to the NKp80 which is an activating receptor of NK cells. Interaction between these two proteins leads to the activation of effector functions of NK cells as well. The aim of this work was the preparation of the recombinant extracellular parts of receptors mNKR-P1CBALB and hAICL, the optimalization of their in vitro refolding and the characterization of proteins using mass spectrometry. The proteins samples will be used for further structural study of the extracellular parts of these leukocytes receptors.
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Production of mouse NK cell receptor NKR-P1C and seeking of tis ligand
Pucholtová, Helena ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
Natural killer or NK cells are immunocytes that mediate innate immunity against pathogens and tumors without pre-exposition to the antigen. They are holding rapid antiviral defense during the initial phase of immune response, before starting the production of antibodies and the development of specific cytotoxic T -lymphocytes. On the surface of NK cells is expressed wide range of inhibition and activation receptors. Important family of those receptors are C - type lectin like from which the family of NKR - P1 ("natural killer cell receptor - protein 1") was discovered first. Diploma thesis deals with the preparation/study of mice NK cell activation receptor NKR- P1C and searching for its binding partner. The soluble form of the protein NKR-P1C was prepared by recombinant expression using the transient transfection of HEK293 cell line (human embryonic kidney 293) with wild type or homogenous glycosylation as IgG - Fc fusion protein, from which was it possible to obtain pure dimer of NKR P1C, after process of affinity purification, TEV protease cleavage and HPLC chromatography. The fusion protein was bound to protein A labeled with a fluorescent probe DyLight 488. Mice tissues and cell lines were labeled by this complex for purpose of seeking ligand.
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Expression of the recombinant extracellular parts of leukocyte receptors AICL a NKR-P1CBALB
Čonka, Martin ; Novák, Petr (advisor) ; Cebecauer, Marek (referee)
v anglickém jazyce NK cells represent a population of lymphocytes which are able to kill certain tumor cells or virally infected cells. The subject of the diploma thesis is a mouse NK cell receptor mNKR- P1CBALB and a human leukocyte receptor hAICL. The mNKR-P1CBALB belongs to the activating receptors and is able to activate the cytotoxic functions of NK cells. The hAICL receptor is a ligand to the NKp80 which is an activating receptor of NK cells. Interaction between these two proteins leads to the activation of effector functions of NK cells as well. The aim of this work was the preparation of the recombinant extracellular parts of receptors mNKR-P1CBALB and hAICL, the optimalization of their in vitro refolding and the characterization of proteins using mass spectrometry. The proteins samples will be used for further structural study of the extracellular parts of these leukocytes receptors.
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Impact of NKR-P1 polymorphism on Ly49 receptors expression in hybrid mouse strains (C57BL/6 x Balb/c, F10-12)
Holubová, Martina ; Fišerová, Anna (advisor) ; Vomastek, Tomáš (referee)
Impact of NKR-P1 polymorphism on Ly49 receptors expression in hybrid mouse strains (C57BL/6 x Balb/c, F10-12) Abstract Natural killer (NK) cells constitute the subpopulation of large granular lymphocytes which mediate spontaneous immune response against infected, transformed or allogeneic cells and thus represent an important component of the innate immunity. NK cells express a wide repertoir of surface receptors which can be either activating or inhibitory and which mediate NK cell recognition and regulation of cytolytic activity. NKR-P1 and Ly49 receptor families belong to the most important murine NK receptors. Both NKR-P1 and Ly49 families are members of C-type lectin-like superfamily of receptors encoded by natural killer gene complex (NKC) on chromosome 6 and include both activating and inhibitory members. The aim of this diploma thesis was to elucidate the impact of Nkr-p1c gene divergence on Ly49 receptors expression and to find out whether the Ly49 and Nkr-p1 gene clusters (which are localized on opposite ends of NKC) are inherited independently or whether the NKC domain is inherited as a complex. The second research interest was to illustrate the influence of the above mentioned divergence on cytotoxic activity of NK cells and tumor growth. In this study, inbred mouse strains C57BL/6 and Balb/c...
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