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Alergije so motnje v delovanju imunskega sistema, s katerimi se dandanes spopada vse več ljudi. Obstajajo sicer različni načini zdravljenja teh bolezni, a so pogosto zahtevni, imajo resne neželene učinke, so neprijetni za pacienta in niso nujno uspešni. Vse to so razlogi, zaradi katerih se raziskovalci usmerjajo v iskanje novih možnih terapij. Velik potencial izkazujejo kratki peptidi-mimotopi, ki lahko posnemajo dejanske alergene in se vežejo na protitelesa IgE pri bolniku, a ne povzročijo alergijske reakcije ter tako sčasoma vodijo v umiritev alergijskega odziva. Odločili smo se jih raziskati v tej magistrski nalogi. Izbrali smo mimotope, ki posnemajo glavni alergen arašidov, saj je prav alergija za arašide ena najpogostejših. Za nosilce smo uporabili mlečnokislinske bakterije – sev Lactococcus lactis, ki veljajo za koristne in so dobro raziskane. Cilj magistrske naloge je bil pripraviti transformirane mlečnokislinske bakterije s sposobnostjo površinskega izražanja mimotopov, ki posnemajo alergen arašidov in se vežejo na protitelesa IgE. Bakterije Lactococcus lactis smo transformirali s plazmidi, ki so vsebovali zapise za izbrane peptide-mimotope, s postopkom elektroporacije. Predhodno smo njihove zapise pomnožili v bakterijah Escherichia coli. Uspešnost transformacije smo preverjali s potrjevanjem pravilnega nukleotidnega zaporedja za izbrane peptide v bakterijah, uspešnost izražanja in funkcionalnost peptidov pa s pomočjo encimsko imunoadsorpcijskega testa (ELISA) ter z metodo prenosa po westernu. S celotnim eksperimentalnim delom smo dosegli postavljeni cilj – proces transformacije je bil uspešen, saj so bakterije na svoji površini izražale želene peptide. To smo dokazali s potrjenimi ustreznimi sekvencami vstavljenega genskega zapisa. S prenosom po westernu smo dokazali prisotnost peptidov na površini L. lactis. Ko smo uporabili humani serum pacientov z alergijo, pa smo s prenosom po westernu potrdili, da so peptidi res mimotopi. Mimotopi imajo velik potencial in upamo, da bodo nadaljnje raziskave prinesle nova, boljša zdravila za alergijske bolezni. Allergies represent a dysfunction in the activity of the immune system and are affecting increasingly more people nowadays. There are different ways of treating these diseases, but these ways are often demanding, have serious side effects, are unpleasant for the patient and not necessarily successful. Due to these reasons, researchers are trying to find new therapy choices. Short chain peptides show a great potential – these are mimotopes, which can mimic allergens and bind with the antibodies IgE of a patient, but do not cause an allergic reaction and in time can appease an allergic reaction. For this master thesis we have chosen mimotopes that mimic the main allergen of peanuts, since peanut allergy is one of the most common allergies. We have used lactic acid bacteria as carriers – strain Lactococcus lactis, which are considered as useful bacteria and are extensively researched. The aim of this master thesis was to prepare transformed lactic acid bacteria that have the ability of surface expression of mimotopes that mimic the allergen of peanuts and bind with the antibodies IgE. Bacteria Lactococcus lactis have been transformed with plasmids, which carried the genes for the chosen peptides – mimotopes - by the process of electroporation. Prior to that process, we have replicated their genes in bacteria Escherichia coli. The success of the transformation was checked/tested with the affirmation of the right sequence of nucleotides for the chosen peptides in the bacteria, while the success of the expression and functionality of peptides was tested with the enzyme-linked immunosorbent assay (ELISA) and the western blot. In the experimental part, we have achieved the set goal – the process of transformation was successful, since the bacteria expressed the peptides on their surface. That was proven with the right sequences of the entered genetic code, while western blot proved the presence of peptides on the surface of L.lactis. In addition, by using the human serum of the patients with allergies and western blot we have proven the fact that peptides are indeed mimotopes. We can then conclude that peptides have a great potential and hope that further research will bring new and improved medicine for allergies. |
