Razvoj rekombinantnih sevov Lactococcus lactis s sposobnostjo dostave proteinskih antagonistov receptorjev za IL-6 in IL-22

Volarič, Nejka (author), Berlec, Aleš (mentor), Plavec, Tina Vida (comentor)

Pogost krivec za razvoj avtoimunskih bolezni in rakavih sprememb je pretirano ali nenadzorovano izražanje citokinov. Citokini so majhne proteinske in glikoproteinske molekule, ki lahko delujejo provnetno ali protivnetno in v telesu opravljajo vlogo komunikacijskega dejavnika med celicami, zlasti pri imunskem odzivu. Dva citokina, ki imata takšno vlogo pri delovanju imunskega sistema, sta tudi IL-6 in IL-22. Z namenom, da bi zavrli njuno pretirano delovanje, smo v okviru magistrske naloge želeli pripraviti genske konstrukte z zapisom za antagoniste njunih receptorjev IL-6Rα in IL-22Rα1. Za zdravljenje avtoimunskih bolezni in raka s pomočjo nevtralizacije citokinskega signaliziranja je trenutno najbolj pogosta uporaba monoklonskih protiteles, ki pa imajo veliko pomankljivosti. Zaradi tega se razvijajo tudi majhni proteinski vezalci neimunoglobulinskega izvora, ki imajo nekatere boljše lastnosti in smo jih zato uporabili tudi v tej magistrski nalogi. Kot gostiteljski organizem in dostavni sistem smo uporabili bakterije Lactococcus lactis, ki zaradi svojih lastnosti lahko preživijo prehod skozi gastrointestinalni trakt. V plazmida pSDBA3b in pSD-Her2-Flag, ki zaradi zapisa za signalni peptid Usp45 in sidrno domeno cAcmA omogočata predstavitev vezalcev na bakterijski površini, smo vstavili gene za neimunoglobulinske vezalce ABR099S, ABR167, NEF108 in NEF163. Vezalce, predstavljene na površini bakterij, smo analizirali z metodo točkovnega nanosa, konfokalno mikroskopijo, z NaDS-PAGE, ki mu je sledil prenos Western (detekcija s protitelesi proti Flag oznaki) ter z metodo pretočne citometrije. Vse metode so potrdile prisotnost vseh vezalcev na površini, pri čemer sta bila v največjem obsegu prisotna Flag-ABR099S in Flag-NEF108. Z metodo NaDS-PAGE in prenosom Western smo potrdili tudi prisotnost vezalcev z oznako Flag v gojišču, kjer so ostali zaradi nepopolne pritrditve sidrne domene na celično steno bakterij po sprostitivi rekombinantnih proteinov iz bakterije. S pretočno citometrijo smo detektirali vezavo proteinov na želene receptorje, ki pa je bila dokaj šibka. Najučinkovitejša je bila vezava proteina Flag-NEF108 na receptor IL-6Rα. Ker je trenutno zelo aktualno tudi področje uporabe postbiotikov, kamor lahko štejemo tudi zunajcelične vezikle, smo preizkusili, če bakterija L. lactis tvori zunajcelične vezikle, in analizirali, če izločeni vezikli vsebujejo rekombinantne proteine. Prisotnost veziklov v vzorcih smo po izolaciji potrdili z elektronskim mikroskopom. Izolirane vezikle smo analizirali z metodo NaDS-PAGE in prenosom po Westernu ter potrdili prisotnost izraženih vezalcev tudi v bakterijskih veziklih.

• Language: Slovenian
• Keywords: Lactococcus lactis, rekombinantni proteini, proteinski vezalci, površinska predstavitev, bakterijski vezikli
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-161669
• Publication date in RUL: 13.09.2024

Secondary language

• Language: English
• Title: Development of recombinant Lactococcus lactis strains with the ability to deliver protein antagonists of IL-6 and IL-22 receptors

Abstract

A common cause of autoimmune diseases and cancer is excessive or uncontrolled expression of cytokines. Cytokines are small protein and glycoprotein molecules that can act pro-inflammatory or anti-inflammatory and play a role as communication factors between cells, especially in the immune response. Two cytokines that play such a role in the immune system are IL-6 and IL-22. To inhibit their excessive activity, this master's thesis aimed to create genetic constructs that would encode antagonists of their receptors IL-6Rα and IL-22Rα1. Currently, the most common approach for the treatment of autoimmune diseases and cancer through neutralization of cytokine signaling is the use of monoclonal antibodies, which, have many limitations. Therefore, small non-immunoglobulin protein binders with some improved properties are being developed, and were also used in this thesis. As an expression host and delivery system, we used bacterium Lactococcus lactis, which can survive the passage through the gastrointestinal tract due to its properties. We inserted genes for non-immunoglobulin binders ABR099S, ABR167, NEF108, and NEF163 into plasmids pSDBA3b and pSD-Her2-Flag, which enable the display of binders on the bacterial surface due to the signal peptide Usp45 and anchor domain cAcmA. We analyzed the binders displayed on the bacterial surface using dot blot, confocal microscopy, NaDS-PAGE followed by Western blot (detection with Flag tag antibodies), and flow cytometry. All methods confirmed the presence of all binders on the surface, with Flag-ABR099S and Flag-NEF108 demonstrating the highest expression. In the next step, we confirmed the presence of Flag-tagged binders in the conditioned medium using NaDS-PAGE and Western blotting. The binders remained in the medium due to incomplete anchoring of the anchor domain in the bacterial cell wall after the secretion of recombinant proteins from the bacteria. Flow cytometry was used to analyze the binding of proteins to the desired receptors, which was relatively weak. The most effective binding was that of the Flag-NEF108 protein to IL-6Rα. Given the current interest in the field of postbiotics, which includes extracellular vesicles, we tested whether L. lactis produces extracellular vesicles and analyzed whether the secreted vesicles contain recombinant proteins. We confirmed the presence of vesicles in samples using electron microscopy. We analyzed the isolated vesicles using NaDS-PAGE and Western blotting and confirmed the presence of expressed binders in bacterial vesicles.

• Keywords: Lactococcus lactis, recombinant proteins, protein binders, surface display, bacterial vesicles