magistrsko delo
Ema Sajovic (Author), Nataša Poklar Ulrih (Mentor), Hrvoje Petković (Thesis defence commission member), Miha Bahun (Co-mentor)

Abstract

Novi betakoronavirus SARS-CoV-2 povzroča koronavirusno bolezen 2019, ki je marca 2020 prerasla v svetovno pandemijo. Protein bodice virusa SARS-CoV-2 je strukturni transmembranski glikoprotein, ki je ključen za vstop virusa v gostiteljsko celico. Virus se s C-terminalno domeno proteina bodice, imenovano receptor vezavna domena RBD, veže na receptor gostiteljske celice. Vezava RBD povzroči konformacijsko spremembo proteina bodice, kar omogoči zlitje virusne ovojnice in gostiteljske celične membrane. V magistrski nalogi smo izrazili gene za rekombinantna proteina RBD izvornega seva virusa SARS CoV-2 in seva SARS-CoV-2 delta plus v bakterijah Escherichia coli. Mutacije, značilne za protein RBD seva delta plus (L452R, T478K in K417N), smo uspešno uvedli v zapis za RBD z enostavno metodo asimetrične verižne reakcije s polimerazo, ki temelji na prekrivajočih koncih. Obe različici proteina RBD sta se v E. coli sintetizirali v obliki netopnih inkluzijskih telesc, ki so se raztopila v pufru z visoko koncentracijo uree. Testirali smo več načinov ponovnega zvijanja denaturiranih proteinov RBD iz inkluzijskih telesc, pri čemer se je kot najuspešnejša metoda izkazala postopna dializa. Preverili smo tudi konformacijsko stabilnost RBD divjega tipa virusa in mutiranega virusa delta plus. Prisotnost sekundarne strukture proteinov smo določili s snemanjem cirkularnega dikroizma v daljnem UV območju, dodatno pa smo konformacijo proteinov ovrednotili tudi z merjenjem fluorescence triptofanskih ostankov. Ugotovili smo, da protein RBD izvornega seva med zamrzovanjem oziroma odmrzovanjem ohrani višjo stabilnost kot različica RBD iz seva delta plus.

Keywords

cirkularni dikroizem;delta plus;Escherichia coli;inkluzijska telesca;NaDS-PAGE;postopna dializa;protein bodice;receptor vezavna domena;SARS-CoV-2;urea;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [E. Sajovic]
UDC: 578.834:602.44:543.544.17:577.112(043.2)
COBISS: 157003523 Link will open in a new window
Views: 41
Downloads: 9
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Other data

Secondary language: English
Secondary title: Production of receptor binding domain of SARS-CoV-2 virus S protein in Escherichia coli
Secondary abstract: The novel SARS-CoV-2 betacoronavirus SARS-CoV-2 causes coronavirus disease 2019, which turned into a global pandemic in March 2020. The spike protein of the SARS-CoV-2 virus is a structural transmembrane glycoprotein that is crucial for viral entry into the host cell. The virus attaches to the host cell receptor with the C-terminal domain of the spike protein called the receptor-binding domain (RBD). The binding of the RBD causes a conformational change in the spike protein, allowing fusion of the viral envelope and the host cell membrane. In the master’s thesis, we expressed genes for recombinant RBD proteins of the original strain of the SARS CoV-2 virus and the SARS-CoV-2 delta plus strain in Escherichia coli bacteria. Mutations specific to the RBD protein of the delta plus strain (L452R, T478K, and K417N) were successfully introduced into the RBD transcript using a simple asymmetric overlap extension polymerase chain reaction. Both versions of the RBD protein were synthesised in E. coli in the form of insoluble inclusion bodies, which were dissolved in a buffer with a high concentration of urea. We tested several methods of refolding denatured RBD proteins from inclusion bodies with step-wise dialysis proving to be the most successful method. We also checked the conformational stability of the RBD of the wild-type virus and the delta plus mutant virus. The presence of the secondary structure of proteins was determined by recording the far UV circular dichroism spectrum, and the conformation of proteins was additionally evaluated by measuring the fluorescence of tryptophan residues. We found that the RBD protein of the original strain maintains higher stability during freezing and thawing than the RBD version from the delta plus strain.
Secondary keywords: circular dichroism;inclusion bodies;receptor binding domain;spike protein;step-wise dialysis;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: 1 spletni vir (1 datoteka PDF (XI, 54, [1] f.))
ID: 19304473