University of Zagreb. Faculty of Science. Department of Chemistry.
Abstract
Virusi zaušnjaka (MuV) i ospica (MeV) uzrokuju istoimene bolesti u ljudi koje se mogu spriječiti cijepljenjem, a dobivaju na značaju i u onkolitičkoj viroterapiji. Cjelovitost i čistoća virusnih čestica osiguravaju učinkovitost i sigurnost ovakvih proizvoda, stoga je njihovo pročišćavanje i koncentriranje izuzetno važno u biotehnologiji, ali i temeljnim istraživanjima. Ciljevi ovog rada su ispitati pročišćavanje MuV-a i MeV-a kromatografijom hidrofobnih interakcija (HIC) i ionske izmjene (IEX), imunoafinitetnom kromatografijom (IAC), ultracentrifugiranjem (UC) i dijafiltracijom (DF), ispitati stabilnost virusa u uvjetima pročišćavanja i čuvanja te istražiti njihove lipidome i proteome. Rezultati pokazuju da IEX nije prikladan za pročišćavanje MuV-a i MeV-a, dok HIC i IAC daju relativno visoke prinose infektivnih virusa (60-70%). Na kromatografske prinose značajno utječe promjenjiva heterogenost početnih virusnih uzoraka. Analiza proteoma virusa potvrdila je prisutnost svih strukturnih virusnih proteina te staničnih proteina aktina, aneksina, ciklofilina A, integrina β1 i moezina, a njihov značaj u virionima tek treba utvrditi.Mumps (MuV) and measles (MeV) virus cause mumps and measles in humans, both preventable by vaccination, but they are gaining importance in oncolytic virotherapy. Viral particle integrity and purity ensure efficacy and safety of such products, therefore their purification and concentration is exceptionally important both in biotechnology and basic research. The aims of this work are to investigate purification of MuV and MeV by hydrophobic interaction (HIC), ion-exchange (IEX) and immunoaffinity chromatography (IAC), ultracentrifugation (UC) and diafiltration (DF), investigate virus stability under used purification and storage conditions and investigate their lipidomes and proteomes. Results deem IEX unsuitable for MuV and MeV purification, while HIC and IAC yield relatively high recoveries of infective viruses (60-70%). Chromatographic yields are significantly influenced by initial virus sample heterogeneity. Analysis of virus proteomes confirmed the presence of all structural viral proteins and cellular proteins actin, annexins, cyclophilin A, integrin β1 and moesin, but their significance in virions remains to be determined
