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Priprava in karakterizacija rekombinantnih enoverižnih fragmentov protiteles proti proteinom LARS, HARS, FUS in MATR3
ID Sotlar, Pia (Avtor), ID Župunski, Vera (Mentor) Več o mentorju... Povezava se odpre v novem oknu

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Izvleček
Rekombinantni enoverižni fragment protitelesa (scFv) je genetska fuzija variabilne domene lahke verige protiteles IgG (VL) in variabilne domene težke verige protiteles IgG (VH). Njihova molekulska masa je od 25 do 30 kDa, kar jim daje določene prednosti, zaradi katerih jih uporabljamo za zdravljenje in diagnosticiranje bolezni. Med te spada amiotrofična lateralna skleroza (ALS), nevrodegenerativna bolezen, ki prizadene zgornje in spodnje motorične nevrone. Med proteine, ki so povezani z ALS, spadata tudi proteina spojen v sarkomu (FUS) in matrin 3 (MATR3). Mutacije v genih FUS in MATR3 med drugimi povzročajo moten transport proteinov med jedrom in citoplazmo, posledica česar je nastanek proteinskih agregatov v citoplazmi. K nastanku teh agregatov in samemu razvoju ALS pa pripomore tudi vpliv aminoacil-tRNA-sintetaz (aaRS), ki so ključne pri procesu translacije. V sklopu diplomskega dela smo želeli z uporabo ekspresijskega sistema E. coli izraziti scFv-je proti proteinom, ki so vpleteni v ALS, Z-FUS-5 in Z-MATR3-4 ter scFv-je proti aminoacil-tRNA-sintetazam, O-LARS-15 in J-HARS-4. Najprej smo kompetentne celice E. coli BL21[DE3] transformirali z vektorji. Izražanje smo nato inducirali z dodatkom IPTG-ja in scFv-je izolirali iz periplazme, saj so vsebovali signalno zaporedje iz proteina zunanje membrane A (ompA). Ker so vsi scFv-ji vsebovali haksahistidinsko oznako, smo za izolacijo uporabili nikljevo afinitetno kromatografijo. Po izolaciji smo scFv-ja Z-FUS-5 in Z-MATR3-4 dodatno okarakterizirali s točkovnim prenosom. Ugotovili smo, da omenjena scFv-ja prepoznata proteina FUS in MATR3 v denaturirani in nedenaturirani obliki.

Jezik:Slovenski jezik
Ključne besede:scFv, amiotrofična lateralna skleroza, aminoacil-tRNA-sintetaze, FUS, MATR3
Vrsta gradiva:Diplomsko delo/naloga
Tipologija:2.11 - Diplomsko delo
Organizacija:FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Leto izida:2024
PID:20.500.12556/RUL-156300 Povezava se odpre v novem oknu
COBISS.SI-ID:196434179 Povezava se odpre v novem oknu
Datum objave v RUL:17.05.2024
Število ogledov:130
Število prenosov:107
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Preparation and characterization of recombinant single-chain variable fragments against proteins LARS, HARS, FUS and MATR3
Izvleček:
Recombinant single-chain antibody fragment (scFv) is a genetic fusion of the variable domain of the light chain of IgG antibodies (VL) and the variable domain of the heavy chain of IgG antibodies (VH). Their molecular mass varies from 25 to 30 kDa, providing them with certain advantages that make them useful for disease treatment and diagnosis. Among these is amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects upper and lower motor neurons. Among the proteins associated with ALS are fused in sarcoma (FUS) and matrin 3 (MATR3). Mutations in FUS and MATR3 genes, among others, disrupt protein transport between the nucleus and cytoplasm, leading to the formation of protein aggregates in the cytoplasm. The development of ALS is also influenced by aminoacyl-tRNA synthetases (aaRS), key enzymes in the translation process. As part of the thesis project, we aimed to express scFv against proteins involved in ALS, specifically Z-FUS-5 and Z-MATR3-4, and scFv against aminoacyl-tRNA synthetases, O-LARS-15 and J-HARS-4, using the E. coli expression system. First competent E. coli BL21[DE3] cells were transformed with vectors. Expression was then induced with the addition of IPTG, and scFvs were isolated from the periplasm, as they contained a signal sequence from outer membrane protein A (ompA). Since all scFvs contained a hexahistidine tag, nickel affinity chromatography was used for isolation. After isolation, scFvs Z-FUS-5 and Z-MATR3-4 were further characterized using dot blotting. We found that the mentioned scFvs recognize the proteins FUS and MATR3 in denatured and native forms.

Ključne besede:scFv, amyotrophic lateral sclerosis, aminoacyl-tRNA synthetase, FUS, MATR3

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