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Razvoj mlečnokislinske bakterije Lactococcus lactis s sposobnostjo dostave proteinskih ligandov receptorja za interlevkin 23 = Development of lactic acid bacterium Lactococcus lactis with the ability to deliver interleukin 23 receptor binding proteins : enoviti magistrski študij farmacijaBenko, Anja, farmacevtkaKronično vnetno črevesno bolezen (KVČB) delimo na ulcerozni kolitis ter Crohnovo bolezen. Za obe obliki je vzrok prekomerna aktivacija imunskega odziva v sluznici črevesja, pri čemer pomembno vlogo ... igra citokinska os IL-23/Th17. Trenutno zdravljenje poteka sistemsko z aminosalicilati, imunosupresivi, kortikosteroidi, pri najhujših oblikah pa s protitelesi proti različnim tarčam v procesu vnetnega odziva. Ker imajo protitelesa visoko ceno, veliko neželenih učinkov ter pri velikem deležu bolnikov niso učinkovita, poteka razvoj majhnih proteinov z visoko afiniteto vezave, specifičnostjo ter sposobnostjo inhibicije vnetja. V okviru magistrskega dela smo uporabili rekombinantne proteine REX009, REX115 in REX125 s potrjenim antagonističnim delovanjem na receptor za IL-23 in jih nadgradili s tarčno dostavo. Kot dostavni sistem smo izbrali mlečnokislinske bakterije Lactococcus lactis, saj imajo status GRAS (Generally recognized as safe) in pripomorejo k vzpostavitvi normalne mikrobiote v sluznici črevesja. Cilj je bil pripraviti bakterije s sposobnostjo izločanja proteinov REX v gojišče in bakterije s sposobnostjo površinske predstavitve proteinov REX. Za pripravo plazmidov z genskimi konstrukti REX smo uporabili plazmide pNZ8148, pSDBA3b in pGEM-T Easy. Končni plazmidi so bili pripravljeni v ogrodju plazmida pSDBA3b, ki omogoča z nizinom nadzorovano izražanje genov, z dodanimi geni za signalni peptid Usp45, označevalni peptid Flag, protein REX ter površinsko sidro cAcmA. V procesu priprave končnih konstruktov smo genske fragmente pomnoževali s PCR, jih ločevali z agarozno gelsko elektroforezo ter jih v plazmide vstavljali z restrikcijo in ligacijo. Plazmide smo analizirali s postopkom PCR na kolonijah in z določanjem nukleotidnega zaporedja ter s preverjenimi transformirali bakterije. Izražanje proteinov REX smo preverili po ločevanju s postopkom NaDS PAGE, in sicer s metodama prenosa po westernu ter z barvanjem z barvilom Commassie Brilliant Blue. Izločanje proteinov REX ter obseg njihove površinske predstavitve smo preverili s pretočno citometrijo. Potrdili smo uspešno vezavo na receptor za IL-23 za vse površinsko predstavljene proteine REX, pri čemer smo opazili najmočnejšo vezavo s proteinom REX125. Ob tem smo ugotovili tudi negativen vpliv sklopitve z označevalnim peptidom Flag na obseg vezave. Dodatno smo opravili modificiran test ELISA, s katerim smo potrdili prisotnost izločenih proteinov REX v gojišču in njihovo sposobnost vezave na imobiliziran receptor IL-23R, pri čemer je največji obseg vezave izkazal protein REX009. V raziskavi smo tako potrdili zmožnost rekombinantnih bakterij za dostavo vezalcev receptorja IL-23R, njihovo terapevtsko učinkovitost pa bo potrebno ovrednotiti na živalskem modelu KVČB.Type of material - master's thesis ; adult, seriousPublication and manufacture - Ljubljana : [A. Benko], 2019Language - slovenianCOBISS.SI-ID - 4863089
Author
Benko, Anja, farmacevtka
Other authors
Berlec, Aleš
Topics
Imunski odziv |
Kronična vnetna črevesna bolezen |
Metapodatki |
Lactococcus lactis |
površinska predstavitev |
rekombinantni proteini |
nadzarovano izražanje genov |
proteini REX |
western prenos
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Library/institution |
City | Acronym | For loan | Other holdings |
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Faculty of Pharmacy, Ljubljana | Ljubljana | FFALJ |
reading room 1 cop.
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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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Benko, Anja, farmacevtka | ![]() |
Berlec, Aleš | 24257 |
Source: Personal bibliographies
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