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Recombinant aspartic proteases of blood-feeding parasites
Váchová, Jana ; Entlicher, Gustav (referee) ; Konvalinka, Jan (advisor)
The blood fluke Schistosoma mansoni and the hard tick Ixodes ricinus produce an aspartic protease cathepsin D which initiates degradation of hemoglobin, their key nutrient. First, in the presented work, the protocol for refolding and activation of the zymogen of cathepsin D from I. ricinus (IrCatD) was developed and optimized. In acidic pH the propeptide of IrCatD zymogen was removed by an auto-activation mechanism. Further, a kinetic assay with fluorogenic substrates was employed to study functional properties of IrCatD including pH optimum, substrate and inhibition specificities. Second, two isoforms of cathepsin D from S. mansoni (SmCatD) were produced using recombinant expression in E. coli. These recombinant proteases were isolated from inclusion bodies using affinity chromatography under denaturating conditions, and protocol for their refolding was developed and optimized. The studied aspartic proteases are pharmacological targets: inhibitors of SmCatD represent potential chemotherapeutics for the treatment of schistosomiasis, and IrCatD is a candidate antigen for the development of novel anti-tick vaccines.
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Molecular characterization of NADPH oxidase in the gut of the tick \kur{Ixodes ricinus}
KUČERA, Matěj
This thesis focuses on characterization of one member of NADPH oxidases - dual oxidase (DUOX) which has been described as the main factor of epithelial immunity in the gut of model organism Drosophila melanogaster. We have identified an orthologous gene coding for DUOX in the tick I. ricinus and described its tissue expression profile. The DUOX is mainly expressed in the gut of unfed ticks and seems to be downregulated upon artificial microbial infection. A fragment of tick DUOX was prepared as recombinant protein and used for preparation of specific antibodies to be used for further characterization of the enzyme. Our main aim is to highlight the importance of tick DUOX producing the reactive oxygen species and their role in the defence against pathogenic organisms within the tick gut.
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Recombinant cysteine peptidases of ticks
Srba, Jindřich
Recombinant cysteine peptidases of Ixodes ricinus Jindřich Srba Abstract Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In a tick, Ixodes ricinus, cysteine proteases, cathepsins B and L, are part of a gut-associated multi-peptidase complex. This thesis deals with preparation of recombinant procathepsins B and L and characterization of functional and biochemical properties of cathepsins B and L. After expression procathepsins B and L in E. coli and their purification from inclusion bodies by affinity chromatography, the basic strategy was searched that would enable to achieve successful refolding. A suitable type in both cases showed to be "basic" refolding. Both refolded procathepsins were auto-activated in the acidic environment of pH 4,0; cathepsin B (but not cathepsin L) also at pH 5,5. Activity was determined at pH dependence substrate specificity and inhibition. The pH optimums for hydrolytic activity of cathepsins B and L were in the range of 4,5 - 5,5 and 3,0 - 3,5, respectively. Both enzymes were blocked by an inhibitor of cysteine peptidase group and each of them by specific inhibitors designed for mammalian cathepsins B and L. The results of this thesis show that cathepsins B and L produced in the gut I. ricinus exhibit exo- and endo-peptidase...
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Characterization of a defensin of the tick \kur{Dermacentor marginatus}
LEŠTINOVÁ, Kateřina
Antimicrobial peptides (AMPs), as a part of innate immune system of ticks and other living organisms, are able to eliminate pathogens. In ticks the most important group of AMPs is defensin family. In this work, defensin from the tick D. marginatus was studied. The defensin gene was isolated from D. marginatus fed females. Using RT-PCR the gene expression was detected in salivary glands and mitgut. Recombinant protein was expressed in the procaryotic expression system, purified and tested for its antimicrobial activity. Specific polyclonal rabbit antibodies (anti DR IgG) were prepared and tested for their specifity and sensitivity.
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Preparation of recombinant inhibitor of serine proteases from the tick \kur{Ixodes ricinus}
VLNOVÁ, Ivana
Tick serine protease inhibitors could be important anti-tick vaccines targets because of their properties and functions. The aim of this work was to prepare recombinant inhibitor of serine proteases from the tick Ixodes ricinus in baculovirus expression system. Two tick saliva proteins of the serpine superfamily were selected for this purpose and transformed into plasmids. One recombinant protein was expressed in baculovirus expression system, purified and its biochemical analyses were done.
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IrAM4: Partial characterisation of a molecule similar to \recke{alpha}\dindex{2}-macroglobulin from a tick \kur{Ixodes ricinus}
ABSOLONOVÁ, Markéta
Ixodes ricinus is a hard tick that can transmit several diseases that are capable of affecting humans. Among those are tick-borne encephalitis and Lyme disease. The focus of this work is on IrAM4, a member of tick ?2-macroglobulin family (?2M-F) of proteins which belong to the evolutionarily oldest constituents of the innate immune system. ?2-macroglobulins are protease inhibitors and act primarily in inactivation of proteases secreted by invading pathogens within their infection cycle. The aim of this study was to identify ?2-M of Ixodes ricinus (IrAM4) from corresponding ortholog of protein ?2-M named IsAM4 present in the genome of closely related Ixodes scapularis. The partial sequence was determined by amplification of cDNA and subsequent sequencing of PCR products. RT-PCR tissue profiling revealed that IrAM4 is present in ovaries and salivary glands but not in the tick gut. The recombinant fragment of IrAM4 was afterwards prepared for immunization of a rabbit and the obtained polyclonal antibodies were used for Western blot analysis. The results showed that IrAM4 is mainly present in the hemolymph and probably in salivary glands and ovaries but it is not expressed in the gut. The native IrAM4 seems to be composed of two disulfide bound subunits. However, the exact structure of the molecule was not analyzed in this work.
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IrAM9 - a member of a thioester-containing protein family from the hard tick \kur{Ixodes ricinus}
RATHNER, Petr
The thioester-containing proteins (proteins of ?2-macroglobulin family) are constituents of innate immunity, which comprise (i) universal protease inhibitors of ?2M type, (ii) C3, C4, C5 components of the complement system, (iii) insect thioester-contaning proteins (iTEPs) and (iv) macroglobulin complement related proteins (MCR). In this work, the partial structure of the thioester-containing protein from the hard tick Ixodes ricinus (IrAM9) which belong to the MCR was determined by sequencing and cloning of PCR products. The IrAM9 message is expressed in the ovaries and salivary glands of partially engorged females. Received recombinant protein was used to get polyclonal serum which was obtained by repeated rabbit immunization. The obtained antibodies were used in an attempt to detect the native protein in tick tissues by immunoblotting method.
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