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Transcriptomic and functional analysis of salivary proteins from the tick \kur{Ixodes ricinus}
CHMELAŘ, Jindřich
This thesis was focused on the identification and characterization of the salivary proteins from Ixodes ricinus, the European vector of Lyme disease and tick-borne encephalitis causative agents. In the first part of this work, the the transcriptomic approach was used in order to identify and describe I. ricinus salivary proteins. The second part is dealing with functional and structural characterization of the salivary protein named IRS-2 (I. ricinus serpin-2).
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Characterization of two members from the multigenic family of one-domain Kunitz-inhibitors from the tick \kur{Ixodes ricinus}
SINGEROVÁ, Barbora
Two new genes encoding proteins Monolaris 1 and Monolaris 2 were isolated from tick Ixodes ricinus. Both cDNA fragments code for 94 aminoacid residues long protein with molecular mass 8,1kDa (Monolaris 1) and 8,3kDa (Monolaris 2). The function of Monolaris 1 was tested by using RNA interference in adult females of Ixodes ricinus that were subsequently fed on guinea-pigs. Body mass, egg mass and mortality were measured to evaluate the effect of gene silencing. Recombinant protein Monolaris 1 was prepared in bacterial expression system and antibodies against this protein were raised by immunization of a rabbit. Antibodies reacted with approximately 190kDa big protein in salivary gland, ovary and gut whereas monomer of Monolaris 1 was not detected in tick saliva, salivary glands and other tissues.
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Expression of Kunitz-type recombinant protein, potential toxin from the salivary glands of the tick \kur{Ixodes ricinus}
CERMANOVÁ, Tereza
Ticks feed on their vertebrate hosts for number of days, providing enough time for development of an effective immune response. To overcome this, tick saliva contains a complex mixture of active substances which mediate host defense mechanisms. Extremely important role at the tick-host interface is played by the protease inhibitors. In this work, we have focused on the protein named Ixocludin 2, a member of the Kunitz type/Bovine trypsin inhibitors of serine proteases, related also to the potassium channel blockers toxins. Three different expression systems (Escherichia coli, Chinese hamster ovary and Pichia pastoris) were tested to prepare an active recombinant Ixocludin 2, out of which, only bacterial system was in part successful.
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Identification and isolation gene encoding peroxiredoxin, detoxification enzyme involved in antioxidant defense of hard tick Ixodes ricinus.
HAVRAN, Jiří
Antioxidant enzymes play an important role in protection against the oxygen radicals generating during aerobic metabolism, as well as in defense against host immune responses. Peroxiredoxin isolated from the hard tick Ixodes ricinus belongs to 1CysPrx group of peroxiredoxins. It is expressed in all stages of hard tick (larva, nymph and adult) and in all tissues examined. The full gene (EU373821) is 756 nt long and consists of 251 amino acids, encoding 27.94 kDa protein. This is the first report of isolation of detoxification enzyme (peroxiredoxin) involved in antioxidant defense of hard tick Ixodes ricinus.
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Characterization of 18,7 and 19 kDa groups of secreted proteins in the salivary glands of the castor bean tick \kur{Ixodes ricinus}
SOUČKOVÁ, Nina
The recombinant protein c90 was prepared and polyclonal antibodies against this protein were raised. The dsRNA was made for the experiments with RNA interference. The samples from dissected tissues of dsRNA silenced ticks were tested by RT-PCR and Western blot. Results suggest that protein c90 plays a role in the tick body during the reaction to injury. Finally, another experiment with injection of water, G+ and G- bacteria into the ticks was realized. It was found that the members of the 18,7 kDa protein family can create multimers. The overexpression of silenced genes was observed during RNAi experiments despite of expected inhibition of c90 production. These results together with the bioinformatics analysis could mean that these proteins are important the physiology of tick probably as a reaction to injury. However c90 protein is produced only in the first phase of feeding which could mean that it has some role in the tick-host interaction as well.
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Ricinusin {--} a new antimicrobial peptide isolated from the hard tick \kur{Ixodes ricinus}: the third member of the tick histidine-rich defense protein family.
DORŇÁKOVÁ, Veronika
New gene encoding antimicrobial protein (ricinusin) was isolated from I. ricinus. The 405 nt cDNA fragment contained a 135 aa ORF, encoding a 14.5 kDa protein with 19 aa signal peptide. Gen Bank comparison of ricinusin showed weak similarity to a new type of histidine rich antimicrobial protein with unique HEAHEAHEA repeat from Boophilus microplus and Amblyomma hebraeum. The differential gene expression was strongly induced by blood feeding in larva, nymph and adults. The site of differential expression in salivary glands and gut indicates that ricinusin is induced as a part of broad-spectrum immune response. To study the amtimicrobial capacity of ricinusin and its capatibility in clearing Borrelia burgdorferi infections, it was expressed as a His-tagged fusion protein in bacterial expression system. Analysis of the genomic organization of ricinusin gene showed that it is intronless.
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Potentials and limits of RNA interference in the tick \kur{Ixodes ricinus}
MUSIL, František
The function of chitin binding protein (CBP) and two isoforms of cathepsin B (cathB1, cathB2) were tested by using RNA interference in the tick I. ricinus. Two different methods have been used to deliver dsRNA for RNAi in ticks {--} injection and capillary feeding. The synthesized dsRNA was used to find out the impact of RNAi in the tick tissues, which were tested by RT-PCR and Western blot. The expression of CBP was successfully silenced by RNAi in the salivary glands. The silencing of cathB1 and cathB2 in the gut was less effective, but still limited tick`s ability to feed.
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