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Pridobivanje ter karakterizacija rekombinantnega egerolizina in proteina z domeno MACPF iz entomopatogene glive Beauveria bassiana.
ID Lemež, Špela (Author), ID Kraševec, Nada (Mentor) More about this mentor... This link opens in a new window

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Abstract
Perforini so proteini, ki s tvorjenjem por v membrane predstavljajo velik biotehnološki potencial na biofarmacevtskih, analitičnih in agronomskih področjih. Znanih je več različnih perforinov in proteinskih parov, ki skupaj tvorijo pore, njihov širok spekter možne uporabe pa kliče po novih raziskavah. Pomembno je natančno poznavanje njihovih molekularnih mehanizmov, ki vplivajo na njihovo biološko funkcijo in možne aplikacije. V tej diplomski nalogi smo se odločili preveriti potencial entomopatogene glive Beauveria bassiana, njenega egerolizina in proteina z domeno MACPF, z namenom ugotavljanja njune interakcije pri formiranju por. Izbrali smo dva gena, za katera je obstajala verjetnost, da glivi omogočata entomopatogeno delovanje s tvorbo por v žuželčjo kutikulo. Tekom raziskave smo uspešno klonirali in izrazili oba proteina v rekombinantni bakteriji, egerolizin smo uspešno izolirali v čisti obliki. Na njem smo uspešno izvedli več analitskih metod, ki so pokazale, da smo protein najverjetneje pridobili v pravilnem zvitju. Sedimentacijski test in površinska plazmonska resonanca sta pokazala močno vezavo egerolizina na kombinacijo lipidov ceramid fosfoetanolamin in sfingomielin, kar nakazuje na predvideno biološko funkcijo. Za natančnejšo določitev bioloških funkcij obeh proteinov in dokončno preverbo njunih potencialov kot pesticidnih sredstev in morebitne druge aplikacije pa so zagotovo potrebne nadaljnje analize.

Language:Slovenian
Keywords:biotehnologija, entomopatogene glive, egerolizini, domena MACPF, perforini
Work type:Bachelor thesis/paper
Typology:2.11 - Undergraduate Thesis
Organization:BF - Biotechnical Faculty
Publisher:[Š. Lemež]
Year:2018
PID:20.500.12556/RUL-101845 This link opens in a new window
UDC:632.937.14:601.4:577.12(043.2)
COBISS.SI-ID:9006969 This link opens in a new window
Publication date in RUL:08.07.2018
Views:1240
Downloads:427
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Secondary language

Language:English
Title:Production and characterisation of aegerolysin and protein with MACPF domain from entomopathogenic fungus Beauveria bassiana.
Abstract:
Perforins are proteins that have a large biotechnological potential in biopharmaceutical, analytic and agronomical fields because of their pore forming abilities. There is already a wide spectre of known perforins and protein couples that jointly form pores, but their wide spectre of potential use calls for new research. Analysis of molecular mechanisms that effect their biological function as well as their potential applications is crucial. In this thesis we decided to discover potential of entomopathogenic fungi Beauveria bassiana, its aegerolylin and protein with MACPF domain with a goal of discovering their interaction in formulation of pores. We selected two genes, for which there was a plausibility, that they are enabling fungi its entomopathogenic activity, with formation of pores into insect cuticle. During the research we successfully cloned and expressed both proteins from recombinant bacterium and successfully isolated aegerolysin in a pure form. On this protein we than preceded to conduct analytical methods, that confirmed, that we most likely gained it in its native form. Sedimentation test and SPR showed strong bonds of aegerolysin to lipid combination of ceramide phosphoethanolamine and sphingomyelin which matches predicted biological function. For exact determination of biological functions of both proteins and final discovery of their potential use as pesticide and other applications further research is needed.

Keywords:biotechnology, aegerolysin, entomopathogenic fungi, MACPF domain, perforins

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