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Synthesis of 5'-UTR of the PSM-E variant of GCPII gene as a standard for RT-PCR
Růžičková, Barbora ; Ingr, Marek (advisor) ; Martínek, Václav (referee)
PCR method has recently become a key element for the syntesis of genes. In this work two-step DNA synthesis based on PCR was used in order to obtain 5'-UTR of the PSM-E splicing variant of glutamate carboxypeptidase II (GCPII) which should serve as a standard for qPCR in real time. Another aim of this work was to clone this sequence into the plasmid PCRII-TOPO PSM-E for the purpose of protein expression in insect cells in the baculovirus expression system. In the first PCR the middle section of the 5'-UTR sequence was synthesized. This product was subsequently amplified in the second PCR using the first and last oligonucleotide. The product of the second PCR was ligated into the plasmid pUC19 and E. coli was transformed by tis construct. Sequence correctness was confirmed by sequencing analysis. Subcloning of the insert from pUC19 to pCRII-TOPO PSM-E was carried out and the sequence was checked by sequencing analysis again. The obtained construct was used as a standard in qPCR in real time in order to determine the concentration of the samples of mRNA from the prostate tissues of oncologic patients
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Synthesis of the Var14 variant of 5'-UTR of GCPII gene as a standard for RT-PCR
Petrovová, Gabriela ; Ingr, Marek (advisor) ; Černá, Věra (referee)
The aim of this work was to prepare the synthetic 5'UTR sequence of splicing variant Var 14 of glutamate carboxypeptidase II (GCPII). A method of two-step PCA was used to this purpose. The sequence was divided into 8 overlapping oligonucleotides that were combined into a single dsDNA by two consecutive PCR. Product of the synthesis was cloned into the auxilliary cloning vector pUC19. After the sequencing analysis detected mutations were corrected. The product was subcloned into the target vector pcDNA4 His Var 14 which already contained the sequence GCPII gene. This construct was then used for the construction of the calibration curve, which will serve as a standard for RT-PCR for quantitative detection of this variant of GCP II in patients with prostate cancer. Construct will be further used as an expression vector to produce of the variants Var 14 GCPII in eucaryotic baculovirus expression system. Keywords: 5'UTR, two-step PCA, pUC19, RT-PCR, GCPII
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Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
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Structural biology of complex of rat NK cell receptors NKR-P1B and Clrb
Dvorská, Anna ; Vaněk, Ondřej (advisor) ; Ingr, Marek (referee)
The Natural Killer (NK) cells have an important role in the nonspecific immunity of the or- ganism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of sti- mulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhi- bition receptor NKR-P1B ("natural killer cell receptor - protein 1B") and its ligand Clrb ("C-type lectin-related ligand b"). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won't be destroyed. If the cell gets infected by the rat cytome- galovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI− ) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors - disulfidic homo- dimers - were prepared as the fusion construct with IgG Fc (using...
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Heterologous expression of human NADPH:cytochrome P450 reductase
Mazurová, Martina ; Martínek, Václav (advisor) ; Ingr, Marek (referee)
Study of carcinogenesis is associated with study of xenobiotics metabolism, which is topic studied in our laboratory. Mixed-function oxygenase system (MFO system) is significantly contributing to the metabolism of xenobiotics. Pure recombinant proteins participating in MFO system are frequently utilized in in vitro metabolic experiments. The heterologous expression method is often used to obtain the pure recombinant enzymes. Heterologous expression was employed to prepare human NADPH:cytochrome P450 oxidoreductase. This membrane enzyme reduces cytochrome P450 and enables its catalytic activity. Vectors with synthetic gene for human NADPH:cytochrome P450 oxidoreductase based on pUC19 and pET22b plasmids were prepared and verified. Recombinant protein was produced in E. coli BL21-Gold and E. coli BL21-CodonPlus-RIL cells. Both cell strains produced high levels of the protein; however the major part of the protein was present predominantly in inclusion bodies. Expression conditions were therefore optimized to obtain higher yields of native protein bound in bacterial membrane fraction. [In Czech]
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Preparation of human NK cell activation receptor NKp80 and its ligand AICL
Kalousková, Barbora ; Vaněk, Ondřej (advisor) ; Ingr, Marek (referee)
NK buňky (z angl. natural killer cells, přirozeně zabíječské buňky) hrají klíčovou roli při rozpoznávání a ničení nádorových, infikovaných nebo jinak pozměněných buněk. Na svém povrchu nemají antigenně specifické receptory, proto je řadíme mezi složky přirozené imunity. K rozpoznání cílových buněk slouží řada jiných povrchových receptorů. Inhibiční receptory zajišťují buněčnou toleranci, naopak aktivační receptory spouští cytotoxické mechanismy vedoucí k apoptóze a tedy lýzi buňky. Díky této vlastnosti jsou NK buňky intenzivně studovány v souvislosti s imunoterapií nádorových onemocnění. Jedním z aktivačních receptorů je NKp80 rozpoznávající svůj ligand AICL. Oba proteiny patří do rodiny receptorů podobných lektinům C-typu. Tento komplex se účastní nejenom přímé lýze maligních buněk myeloidního charakteru, ale má také důležitou roli v imunomodulaci zánětu. Předmětem této diplomové práce je příprava receptoru NKp80 a jeho ligandu AICL. Receptor NKp80 byl připraven v linii lidských embryonálních ledvinných buněk (HEK 293S GnTI- ). Byly připraveny stabilně transfekované linie produkující protein NKp80 konstitutivně nebo indukovatelně. Zapojení disulfidických můstků a obsazení N-glykosylačních míst proteinu NKp80 bylo ověřeno hmotnostní spektrometrií. Dále byl optimalizován postup Mgr. Jiřího Nového na...
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Structural biology of complex of rat NK cell receptors NKR-P1B and Clrb
Dvorská, Anna ; Vaněk, Ondřej (advisor) ; Ingr, Marek (referee)
The Natural Killer (NK) cells have an important role in the nonspecific immunity of the or- ganism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of sti- mulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhi- bition receptor NKR-P1B ("natural killer cell receptor - protein 1B") and its ligand Clrb ("C-type lectin-related ligand b"). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won't be destroyed. If the cell gets infected by the rat cytome- galovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI− ) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors - disulfidic homo- dimers - were prepared as the fusion construct with IgG Fc (using...
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Study of the cleavage kinetics of Gag polyprotein from HIV-1 virus by the viral proteinase
Krištofičová, Ivica ; Ingr, Marek (advisor) ; Martínek, Václav (referee)
Gag polyprotein is the precursor of HIV-1 structural proteins, required for correct assembly, budding and maturation of viral particle within HIV-1 life cycle. The process of maturation into an infectious virion is dependent on Gag and GagPol cleavage at nine predefined sites by HIV-1 proteinase. Its disruption is one of the main targets of HIV treatment. HIV-1, however, develops resistance to the proteinase inhibitors by creating mutations in both the proteinase and the substrate. The Gag processing by HIV-1 proteinase is a highly sequential process, that happens in specific order and rate. Previous biochemical studies determined the kinetic data of these processes using oligopeptides representing naturally occuring cleavage sites. This thesis describes the cleavage of the Gag polyprotein itself, which is the natural substrate of HIV-1 proteinase. For this purpose, the full-length Gag polyprotein was recombinantly prepared in bacterial expression system. The cleavage was carried out and its products were analyzed via SDS-PAGE and Western blotting. The substrate specificity of the wild-type and mutant HIV-1 proteinase with respect to the full-length wild-type Gag polyprotein was compared. Substantial differences were observed between the rates of individual steps of cleavage by the wild-type and mutant...
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