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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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Cloning and expression of a portion of \kur{Bombyx Mori Ser/2} gene
KONÍKOVÁ, Tereza
A 897 bp section of Bombyx mori Ser-2 gene was amplified, cloned into a bacterial expression vector, and used to prepare a recombinant sericin-like protein. The protein is expected to contain domain supporting growth of mammalian cells. It will be used in assays verifying this assumption; if confirmed, recombinant protein will be considered for preparations of scaffold guiding tissue reconstruction.
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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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Studies on the sericin 3 of \kur{Bombyx mori} and cloned sericin into \kur{Escherichia coli}
KRŮČEK, Tomáš
The spun-out silk fiber consists of two fibroin filaments that are cemented together by sericin coating. The serine-rich sericins, which make 20-30% of the cocoon silk proteins in Bombyx mori, are dissolved in hot water during silk fiber reeling from the cocoon. The sericin extract is usually discarded. Only small amounts are currently used in cosmetics and lately also as replacement of bovine serum products in the cell culture media. The use in culture media is hindered by poor standardization of the extracts. To overcome this problem, we attempted preparation of several recombinant proteins based on partial sequences of one out of the three sericin genes of Bombyx mori, sericin 3. Translation product of the Ser3 gene is extremely serine-rich with a relatively high representation of the aspartate, asparagin, glutamate, and glutamine. Using specific primers we have prepared a cDNAs of 267, 279, 525, 672, and 528 bp, respectively, derived from the Ser3 gene. The cDNAs were cloned and expressed as fusion proteins with hexahistidine in Escherichia coli. The proteins were purified by affinity chromatography and analyzed by acrylamide electrophoresis. Ser3 gene contain repetitive motifs rich in serine and including some charged amino acids.
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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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