Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen

Geeniterapiassa sairautta hoidetaan siirtämällä soluihin siirtogeeni, joka koodaa haluttua proteiinituotetta. Virukset toimivat hyvinä geenien kuljettimina eli vektoreina, sillä niille on luontaista siirtää perimänsä kohdesolun replikoitavaksi. Adenovirus on hyvin yleisesti käytetty geeniterapiavekt...

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Main Author: Lepola, Saana
Other Authors: Matemaattis-luonnontieteellinen tiedekunta, Faculty of Sciences, Bio- ja ympäristötieteiden laitos, Department of Biological and Environmental Science, Jyväskylän yliopisto, University of Jyväskylä
Format: Master's thesis
Language:fin
Published: 2019
Subjects:
Online Access: https://jyx.jyu.fi/handle/123456789/64998
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author Lepola, Saana
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_facet Lepola, Saana Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä Lepola, Saana Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_sort Lepola, Saana
datasource_str_mv jyx
description Geeniterapiassa sairautta hoidetaan siirtämällä soluihin siirtogeeni, joka koodaa haluttua proteiinituotetta. Virukset toimivat hyvinä geenien kuljettimina eli vektoreina, sillä niille on luontaista siirtää perimänsä kohdesolun replikoitavaksi. Adenovirus on hyvin yleisesti käytetty geeniterapiavektori, sillä sen ominaisuudet tunnetaan hyvin ja se pystyy infektoimaan monenlaisia soluja eri solusyklin vaiheissa. Adenoviruksen rakenneproteiini IX (pIX) sijaitsee heksoniproteiinien välissä stabiloiden kapsidin rakennetta. Tutkimukset ovat kuitenkin osoittaneet, että pIX:llä on mahdollisesti myös muunlaisia rooleja adenovirusinfektion aikana. Tässä tutkimuksessa oli tarkoitus selvittää, miten kahden genomiltaan erilaisen adenovirusvektorin stabiilisuudet ja infektiivisyydet eroavat toisistaan. Sen lisäksi selvitettiin, voidaanko proteiini IX:n määrää lisätä adenoviruksen kapsidissa yliekspressoimalla sitä tuottosoluissa ja vaikuttaako määrä adenoviruksen kestävyyteen. Molempia vektoreita tuotettiin sekä pIX:n yliekpression kanssa että ilman. Proteiini IX:n lisäämisen vaikutusta adenoviruksen kapsidin kestävyyteen tutkittiin käsittelemällä tuotettuja viruksia eri tavoin ja havainnoimalla erot infektiivisyydessä. Tuotetut virukset puhdistettiin ultrasentrifugoimalla ja dialyysilla. Puhtautta ja pIX:n määrää arvioitiin Coomassie-värjäyksen sekä western blottingin avulla. Lisäksi virusten tiitterit määritettiin ddPCR:n (digital droplet polymerase chain reaction) avulla. Tuotettujen virusten infektiivisyys määritettiin infektoimalla tuottosoluja 2 tunnin ajan ja värjäämällä solut 48 tunnin kuluttua anti-pIX-vasta-aineella. Tulokset luettiin virtaussytometrilla. Eri vektorien stabiilisuuksissa ei nähty tilastollisesti merkittäviä eroja yhdessäkään käsittelykokeessa. Työssä kuitenkin havaittiin, että vektorit, jotka oli tuotettu pIX:n yliekpression kanssa olivat infektiivisempiä kuin ne, jotka oli tuotettu normaalisti tuottosoluissa. Tuloksia voidaan käyttää hyväksi suunniteltaessa tehokkaampia ja kestävämpiä geeniterapiavektoreita lääketeollisuuden käyttöön. In gene therapy genes are used to treat or cure illnesses. These transgenes are delivered into patient's cells with a special tool, vector. Viruses are often used as vectors because of their natural ability to transfer their genes into target cells. Adenovirus is a very common vector in gene therapy because its features are well known and it can infect many different types of cells in various times in cell cycle. Adenovirus structural protein IX (pIX) is located between hexon proteins stabilizing the structure of adenoviral capsid. Studies indicate that pIX might also have additional roles during adenovirus infection. In this study the object was to find out how the stabilities and infectivities of two different adenoviral vectors differ from each other. The aim was also to find out if the amount of pIX can be increased in adenoviral capsid by overexpressing it in the producer cells and if this has an effect to the stability of the capsid. Both vectors were produced with and without pIX overexpression. The effects of pIX overexpression to stability were studied by treating the viruses in different ways and observing the differences in infectivities. Produced vectors were purified using ultracentrifugation and dialysis. The purity and amount of pIX were estimated using Coomassie staining and Western blot analysis. Virus titers were determined using ddPCR (digital droplet polymerase chain reaction). The infectivities were determined by infecting producer cells for 2 hours and staining the cells after 48 hours with anti-pIX antibody. Results were analyzed using flow cytometry. Although statistically significant differences couldn't be observed in stability studies, the viruses which were produced with overexpression of pIX were more infectious than the ones that had been produced in normal producer cells. These results can be utilized when designing more effective and durable viral vectors for the field of gene therapy.
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spellingShingle Lepola, Saana Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen Solu- ja molekyylibiologia Cell and molecular biology 4013 adenovirukset virukset proteiinit geeniterapia geenit tuotto
title Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen
title_full Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen
title_fullStr Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen
title_full_unstemmed Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen
title_short Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen
title_sort proteiini ix n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen
title_txtP Proteiini IX:n yliekspression vaikutus adenovirusvektoreiden stabiilisuuteen ja infektiivisyyteen
topic Solu- ja molekyylibiologia Cell and molecular biology 4013 adenovirukset virukset proteiinit geeniterapia geenit tuotto
topic_facet 4013 Cell and molecular biology Solu- ja molekyylibiologia adenovirukset geenit geeniterapia proteiinit tuotto virukset
url https://jyx.jyu.fi/handle/123456789/64998 http://www.urn.fi/URN:NBN:fi:jyu-201907053584
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