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

ILGOVÁ, Jana, Lucie JEDLIČKOVÁ, Hana DVOŘÁKOVÁ, Libor MIKEŠ, Milan GELNAR and Martin KAŠNÝ. Cystatin: the cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea). In 4th Workshop of European Centre of IchthyoParasitology. 2015. ISBN 978-80-210-8016-4.

Basic information

• Original name: Cystatin: the cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea)
• Authors: ILGOVÁ, Jana, Lucie JEDLIČKOVÁ, Hana DVOŘÁKOVÁ, Libor MIKEŠ, Milan GELNAR and Martin KAŠNÝ.
• Edition: 4th Workshop of European Centre of IchthyoParasitology, 2015.

Other information

• Original language: English
• Type of outcome: Conference abstract
• Field of Study: 10600 1.6 Biological sciences
• Country of publisher: Czech Republic
• Confidentiality degree: is not subject to a state or trade secret
• Organization unit: Faculty of Science
• ISBN: 978-80-210-8016-4
• Keywords in English: Eudiplozoon nipponicum; cystatin; recombinant protein

Abstract

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 synthesized by this taxonomic group. Among important molecules of 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 regulation of blood digestion process. 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 gene coding cystatin of E. nipponicum was expressed in heterologous E. coli (BL21) system using pET19b expression vector. IPTG induced expression (22 °C, 6 hours) was analyzed on SDS-PAGE and soluble recombinant cystatin of E. nipponicum was purified on Ni-NTA agarose column. Production of cystatin was verified by both mass spectrometry (Orbitrap) and Western Blot using His-tag specific antibodies. Activity of recombinant inhibitor was tested by inhibitory assay using recombinant E. nipponicum cathepsin L and fluorogenic substrate Z-FR-AMC. Soluble recombinant cystatin was used for immunization of ICR mice. The polyclonal anti-cystatin sera reacted specifically with the crude extract of E. nipponicum and recombinant cystatin of E. nipponicum (Western blot).

Links

• GAP506/12/1258, research and development project: Name: Interakce hostitel-parazit u krevsajících diplozoidních monogeneí: Výzkum vysoce specializovaných adaptací k parazitismu

Investor: Czech Science Foundation

• GBP505/12/G112, research and development project: Name: ECIP - Evropské centrum ichtyoparazitologie

Investor: Czech Science Foundation

• MUNI/A/1484/2014, interní kód MU: Name: Analýzy diverzity biologických systémů různých úrovní a na různých škálách terestrického a akvatického prostředí (Acronym: BIDA4)

Investor: Masaryk University, Category A