magistrsko delo
Manca Vertot (Avtor), David Stopar (Recenzent), Anja Klančnik (Mentor), Jerica Sabotič (Komentor)

Povzetek

Polisaharidi kapsule bakterij Campylobacter jejuni so pomembni virulentni dejavniki in lahko tvorijo komponente cepiv. Poznavanje popolne strukture in funkcije le-teh je izredno velikega pomena, zato smo v magistrski nalogi želeli vpeljati metodo izolacije polisaharidov kapsule in pridobljene polisaharide kapsule okarakterizirati z jedrsko magnetno resonanco (NMR). Za izolacijo polisaharidov kapsule smo uporabili encimsko metodo in metodo z vročim fenolom, in s slednjo smo pridobili večjo količino vzorca CPS. Kontaminacijo vzorcev s proteini in nukleinskimi kislinami smo preverili s spektrofotometrijo, agarozno elektroforezo in metodo NaDS-PAGE. Ugotovili smo, da polisaharidi kapsule, izolirani z metodo z vročim fenolom, vsebujejo večji delež nečistoč, kot polisaharidi kapsule, izolirani z encimsko metodo. Za detekcijo polisaharidov kapsule in ostalih glikokonjugantnih molekul smo izvedli metodo NaDS-PAGE z barvanjem s srebrom in perjodno kislino ter Schiff-ovim reagentom. Pri fenolnih vzorcih smo določili lipooligosaharide in polisaharide kapsule, medtem ko smo pri encimskih vzorcih opazili le lise pri majhni molekulski masi, ki predstavljajo lipooligosaharide. Pridobljene polisaharide smo okarakterizirali z metodo NMR, kjer smo pri encimskih vzorcih uspešno potrdili prisotnost osnovnih sladkornih komponent polisaharidov kapsule bakterij C. jejuni, ki so skladne z objavljenimi podatki. Prav tako, številni signalni na intervalu anomerne regije nakazujejo na prisotnost različnih oblik polisaharidov kapsule znotraj vzorca. Glede na rezultate lahko zaključimo, da smo optimizirali novo metodo izolacije polisaharidov kapsule bakterij C. jejuni.

Ključne besede

Campylobacter jejuni;zunajcelični polisaharidi;kapsula;jedrska magnetna resonanca;NMR;izolacija polisaharidov kapsule;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL BF - Biotehniška fakulteta
Založnik: [M. Vertot]
UDK: 579.233:543.635.25.054:57.088
COBISS: 87091971 Povezava se bo odprla v novem oknu
Št. ogledov: 226
Št. prenosov: 36
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: ǂThe ǂextracellular polysaccharide layer of Campylobacter jejuni
Sekundarni povzetek: The capsular polysaccharides produced by Campylobacter jejuni are known to be important virulence factors and could be important components of vaccines. Determination of their complete structure and function is extremely important. Therefore, in this master thesis, we present a method for the isolation of capsular polysaccharides and their characterization by nuclear magnetic resonance (NMR). For the isolation of capsular polysaccharides, we used the enzyme method and the hot phenol method, with a higher amount of capsular polysaccharides obtained by the latter. Samples were analysed for contaminating proteins and nucleic acids by spectrophotometry, agarose electrophoresis, and SDS-PAGE. We found that capsular polysaccharides obtained by the hot phenol method contained a higher amount of contaminants than capsular polysaccharides obtained by the enzyme method. Silver and periodic acid-Schiff staining were performed to detect capsular polysaccharides and other glycoconjugates. In the phenol samples, we detected lipooligosaccharides and capsular polysaccharides, whereas in the enzyme samples we observed only low molecular weight bands, indicating the presence of lipooligosaccharides. The structure of the polysaccharides was analysed by NMR. In the enzyme samples, we were able to detect and confirm the presence of sugar monomers of capsular polysaccharides of C. jejuni, which are in accordance with the published data. In addition, we observed multiple peaks in anomeric region, suggesting that multiple forms of capsular polysaccharides exists within the sample. Based on our results, we can conclude that we have optimized a novel method to isolate capsular polysaccharides from C. jejuni.
Sekundarne ključne besede: Campylobacter jejuni;extracellular polysaccharide;capsule;nuclear magnetic resonance;NMR;capsular polysaccharides isolation;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 0
Komentar na gradivo: Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Strani: X, 60 f.
ID: 14040760