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Kloniranje in izražanje bakteriofagnih endolizinov ter proteinskih vezalcev toksinov Clostridioides difficile = Cloning and expression of bacteriophage endolysins and protein binders of Clostridioides difficile toxins : univerzitetni študijski program KozmetologijaCelan, ZalaZdravljenje okužbe z bakterijo Clostridioides difficile zaradi odpornosti proti antibiotikom predstavlja veliko problematiko v sodobnem svetu. Do okužb prihaja zlasti v bolnišničnem okolju ob uporabi ... antibiotikov, ki izzovejo spremembe v sestavi črevesne mikrobiote. C. difficile povzroča vnetja in krvavitve iz debelega črevesa (kolitis), ki vodijo v hudo obliko driske in se lahko končajo tudi s smrtjo. Poglavitna virulentna dejavnika C. difficile sta eksotoksina TcdA in TcdB, glukoziltransferazi, ki delujeta na GTPaze iz družine Rho, s čimer oslabita tesne in fokalne stike v sluzničnem epiteliju. Rekombinantni probiotiki predstavljajo bioalternativni protimikrobni pristop za zdravljenje okužbe s C. difficile in so tematika mnogih (pred)kliničnih raziskav. Okužbo s C. difficile bi lahko zmanjšali ali preprečili s kombinacijo uporabe bakteriofagnih endolizinov, selektivnih do C. difficile, in specifičnih proteinskih vezalcev toksinov TcdA ter TcdB, ki bi se izražali na površini rekombinantne probiotične bakterije Lactococcus lactis. V diplomski nalogi smo se osredotočili na pridobitev vezalcev toksinov (iz skupin afimerov in DARPinov; samih ali v fuziji s C-končno domeno sidrnega proteina AcmA, ki omogočajo vezavo na celično steno L. lactis,) ter endolizina z uporabo ekspresijskega sistema Escherichia coli. V prvem delu naloge smo pripravili ekspresijske vektorje: v modificiran plazmid pET28c (pET28c-HTF), smo vstavili sintezne gene za izbrane proteine. Ekspresijski sev E. coli NiCo21(DE3) smo transformirali s plazmidi in izvedli testno izražanje rekombinantnih proteinov po indukciji z IPTG ter s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata kvalitativno ocenili količino rekombinantnih produktov. Ugotovili smo, da vezalci eksotoksinov v fuziji z AcmA niso topni. Na podlagi rezultatov smo za izražanje v povečanem obsegu izbrali le topne proteine (tj. vezalce eksotoksinov brez AcmA in endolizina). V drugem delu diplomske naloge smo rekombinantne proteine izolirali s kovinsko-kelatno afinitetno kromatografijo ter jih koncentrirali z ultrafiltracijo. Ugotovili smo, da se fuzijski proteini vezalcev eksotoksinov TcdA/TcdB s sidrno domeno AcmA nahajajo v netopni frakciji lizata in se pri uporabljenih pogojih izražanja odlagajo v inkluzijska telesca.Type of material - undergraduate thesis ; adult, seriousPublication and manufacture - Ljubljana : [Z. Celan], 2023Language - slovenianCOBISS.SI-ID - 173734915
Author
Celan, Zala
Other authors
Bratkovič, Tomaž, farmacevt, 1978- |
Bozovičar, Krištof
Topics
Bakterijska rezistenca |
Rekombinantni proteini |
afimeri |
Clostridioides difficile |
čiščenje |
DARPini |
endolizini |
izražanje |
vezalci eksotoksinov
Library | Call number – location, accession no. ... | Copy status |
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Faculty of Pharmacy, Lj. | Knjižnica UNK 615 CELAN ZALA Kloniranj 305 IN: 0017918 |
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Database name | Field | Year |
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Links to authors' personal bibliographies | Links to information on researchers in the SICRIS system |
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Celan, Zala | |
Bratkovič, Tomaž, farmacevt, 1978- | 23598 |
Bozovičar, Krištof | 53738 |
Source: Personal bibliographies
and: SICRIS
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