Genski elektroprenos DNA-cepiva proti antigenom virusa SARS-CoV-2 v mišje fibroblaste L929

Jakopič, Helena (author), Čemažar, Maja (mentor), Žnidar, Katarina (comentor)

Cepiva na osnovi nukleinskih kislin, kot je DNA-cepivo, so ena izmed možnosti cepljenja proti bolezni covid-19. Z uporabo metode genskega elektroprenosa izboljšamo slabo imunogenost DNA-cepiv in zagotovimo boljšo učinkovitost. Pripravili smo cepivo na osnovi plazmidne DNA, z zapisoma za antigena virusa SARS-CoV-2, in sicer za protein bodice (S) in protein nukleokaspide (N). Transfekcijo plazmidne DNA v celice mišjih fibroblastov L929 smo izvedli z genskim elektroprenosom. V in vitro študiji smo preverili izražanje antigenov na ravni mRNA in proteinov. Z uporabo metode RT-qPCR smo dokazali povečano izražanje genov za protein S in N v poskusnih skupinah S, N in N+S. Z metodo ELISA smo dokazali protein N v celičnem mediju in celičnem lizatu. V skupini N+S smo v primerjavi s skupino N v celičnem mediju dokazali šestkrat večjo koncentracijo proteina N. V celičnem lizatu nam je uspelo dokazati trikrat večjo koncentracijo proteina N v poskusni skupini N+S v primerjavi s skupino N. Proteina S s testom ELISA nismo uspeli zaznati ter ga tako nismo potrditi na proteinski ravni. Rezultate in povzetke in vitro študije je Oddelek za Eksperimentalno onkologijo, OI v nadaljevanju uporabil za pripravo in vivo študije na mišjih modelih.

• Language: Slovenian
• Keywords: genski elektroprenos, DNA-cepiva, COVID-19 cepivo, SARS-CoV-2, mišji fibroblasti, in vitro
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publisher: [H. Jakopič]
• Year: 2023
• PID: 20.500.12556/RUL-145123
• UDC: 577.2
• COBISS.SI-ID: 149645827
• Publication date in RUL: 07.04.2023

JAKOPIČ, Helena, 2023, Genski elektroprenos DNA-cepiva proti antigenom virusa SARS-CoV-2 v mišje fibroblaste L929 [online]. Master’s thesis. H. Jakopič. [Accessed 30 March 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=145123

Secondary language

• Language: English
• Title: Gene electrotransfer of a DNA vaccine against SARS-CoV-2 viral antigens in L929 murine fibroblast cells

Abstract

Nucleic acid-based vaccines, such as DNA vaccine, are one of the options for vaccination against the disease COVID-19. Using the Gene Electro Transfer method, we improve the poor immunogenicity of the DNA vaccine and provide better efficacy. We have produced a vaccine based on plasmid DNA. As antigens were used two plasmids that encode SARS-CoV-2 spike (S) or nucleocapsid (N) protein. Transfection of mouse fibroblast cells L929 was performed by Gene Electro Transfer. In in vitro study, we checked the expression of the antigens at the mRNA and protein levels. Using RT-qPCR, we have shown that in experimental groups S, N, and N+S, gene expression for N and S proteins occurs. Finally, antigens were determined at the protein level. N protein was detected by ELISA in the cell medium and cell lysate. In the N+S group in the cell medium, we detected a 6 times higher concentration of N protein than in the N group. In cell lysate, we were able to demonstrate a 3-fold higher concentration of N protein in the N+S experimental group compared to the N group. We were not able to detect S protein with the ELISA assay, thus, could not confirm it at the protein level. The final outcomes of the in vitro study were used by the Department of Experimental oncology, OI for in vivo studies.

• Keywords: Gene electrotransfer, DNA vaccination, COVID-19 vaccine, SARS-CoV-2 virus, mouse fibroblast L929, in vitro