This work is part of a larger project concerning the development of a system to validate therapeutic strategies aimed to blocking the early stages of the replicative cycle of humans pathogenic viruses, in particular interfering with the recognition between cellular receptor and viral anti-receptor. This thesis focuses on the development of recombinant lentiviral particles that incorporate into their envelope the Spike glycoprotein of the pandemic virus SARS-CoV-2 (pseudotyping) in order to mimic the early stages of the replicative cycle of SARS-CoV-2 and the genome expresses a reporter gene to evaluate the efficiency of the pseudotyped particles to infect target cells. The advantage of this system is that it allows us to work in BLS 2 laboratories, while the wild-type virus should be used in BLS 3 laboratories. This thesis work involved the production of pseudotyped lentiviral-like particles with the SARS-COV-2 Spike by transfection of 293T cells which were then used to transduce cells susceptible to SARS-CoV-2 infection and the percentage of cells expressing the reporter protein was assessed by cytofluorometer. The data obtained indicate that this system works and may be used to evaluate molecules that inhibit SARS-CoV-2 entry.
Questo lavoro di tesi si inserisce all’interno di un progetto più ampio riguardante lo sviluppo di un sistema per validare strategie terapeutiche mirate a bloccare le fasi iniziali del ciclo replicativo di virus patogeni per l’uomo, in particolare interferendo con il riconoscimento tra recettore cellulare e antirecettore virale. Questa tesi è incentrata sullo sviluppo di particelle lentivirali ricombinanti che incorporano nel proprio envelope la glicoproteina Spike del virus pandemico SARS-CoV-2 (pseudotipizzazione) al fine di mimare le fasi iniziali del ciclo replicativo di SARS-CoV-2 mentre il genoma esprime un gene reporter per valutare l’efficienza delle particelle pseusotipizzate di infettare le cellule target. Il vantaggio di questo sistema è che ci permette di lavorare in laboratori di BLS 2, mentre il virus wild-type andrebbe utilizzato in laboratori BLS 3. Questo lavoro di tesi ha previsto la produzione delle particelle simil lentivirali pseudotipizzate con la Spike di SARS-COV-2 mediante trasfezione di cellule 293T le quali sono poi state utilizzate per trasdurre cellule suscettibili di infezione da SARS-CoV-2 e la percentuale di cellule esprimenti la proteina reporter è stata valutata al citofluorimetro. I dati ottenuti indicano che questo sistema funziona e potrà essere impiegato per valutare molecole inibitrici l’ingresso del virus SARS-CoV-2.
Sviluppo di un sistema basato su particelle simil lentivirali esprimenti la glicoproteina "Spike" dell'envelope di SARS-CoV-2
CASAGRANDE, MAURO
2021/2022
Abstract
This work is part of a larger project concerning the development of a system to validate therapeutic strategies aimed to blocking the early stages of the replicative cycle of humans pathogenic viruses, in particular interfering with the recognition between cellular receptor and viral anti-receptor. This thesis focuses on the development of recombinant lentiviral particles that incorporate into their envelope the Spike glycoprotein of the pandemic virus SARS-CoV-2 (pseudotyping) in order to mimic the early stages of the replicative cycle of SARS-CoV-2 and the genome expresses a reporter gene to evaluate the efficiency of the pseudotyped particles to infect target cells. The advantage of this system is that it allows us to work in BLS 2 laboratories, while the wild-type virus should be used in BLS 3 laboratories. This thesis work involved the production of pseudotyped lentiviral-like particles with the SARS-COV-2 Spike by transfection of 293T cells which were then used to transduce cells susceptible to SARS-CoV-2 infection and the percentage of cells expressing the reporter protein was assessed by cytofluorometer. The data obtained indicate that this system works and may be used to evaluate molecules that inhibit SARS-CoV-2 entry.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/32839