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Cystatin: the cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea)

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Rok publikování 2015
Druh Konferenční abstrakty
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Popis Eudiplozoon nipponicum (Monogenea, Diplozooidae) typically inhabiting the gills of carp (Cyprinus carpio) is widely distributed Eurasian blood-feeding ectoparasite. The foregoing research of diplozoid Monogenea has been predominatly oriented towards understanding morphology and ecology of particular species. Nevertheless there is a lack of information regarding functional molecules produced by this taxonomic group. Among important molecules produced by various parasitic species belong cystatins (inhibitors of cystein peptidases). Besides regulation of endogenous processes in parasite bodies they play a substantial role e.g. in manipulation of the host immune system and/or blood digestion. The aim of our experimental work is to reveal the presence, structure and function of E. nipponicum cystatin DNA/protein molecules using bioinformatic, molecular and proteomic methods. The transcriptomic data of E. nipponicum was screened for the presence of cystatin sequences. The partial nucleotide sequence was identified, amplified, completed using 3’and 5’ RACE PCR, cloned and sequenced. Gene coding 98 amino acid cystatin of Eudiplozoon nipponicum with predicted molecular weight 10,85 kDa and theoretical pI 6,27 was inserted into pET19b expression vector a cloned into E. coli competent cells (BL 21 strain). Expression was induced by IPTG at 37 °C. The production of recombinant cystatin by E. coli host cells was analysed on SDS-PAGE (Coomasie staining) and verified by mass spectrometry (Orbitrap). Recombinant protein was produced in the insoluble form - incorporated into inclusion bodies. The cystatin from E. nipponicum will be subsequently solubilized and further tested for its functional and structural properties.
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