Production, purification and characterization of Coxsackievirus B1 virus-like particles
Andrejeff, Tanja (2017)
2017
Bioteknologian tutkinto-ohjelma - Degree Programme in Biotechnology
Lääketieteen ja biotieteiden tiedekunta - Faculty of Medicine and Life Sciences
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Hyväksymispäivämäärä
2017-06-29
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:uta-201707172222
https://urn.fi/URN:NBN:fi:uta-201707172222
Tiivistelmä
Background and aims: Virus-like particles (VLPs) are promising vaccine candidates, usually eliciting broad and strong immune responses in humans. The aim of this study was to produce VLPs for coxsackievirus B1 (CVB1). CVB viruses cause severe morbidity and mortality worldwide, causing a major impact on the health care system, but there are no treatments or vaccines available against CVBs. The specific aims of this study were to find out the most efficient insect cell-line for CVB1-VLP production, and finding a scalable purification method for CVB1-VLP and finally the characterization of CVB1-VLP.
Methods: Baculovirus-insect cell expression system was used in the production of CVB1- VLPs. Various steps in the CVB1-VLP production were optimized including the type of insect cell line and the flashBAC DNA variant, the Multiplicity Of Infection (MOI) used for CVB1-VLP amplification, baculovirus cultivation time, type of cell growth medium and culture volumes and the CVB1-VLP purification method. VLPs were concentrated either using tangential flow filtration or PEG-precipitation and purified using multi-step ion exchange chromatography (IEX) purification. The quality and the purity of the CVB1- VLPs were estimated with SDS-PAGE, Western Blotting, dynamic light scattering and Transmission Electron Microscopy analyzes.
Results: The highest CVB1-VLP production level was obtained in High Five cells and they expressed two times more extracellular VLP than Sf9 cells. FlashBAC ULTRA and flashBAC GOLD expressed considerably more extracellular VLP than flashBAC or flashBAC PRIME in High Five cells. Further, the production levels of flashBAC ULTRA and flashBAC GOLD were almost equal. MOI 1 seemed to give the highest production levels. Highest yield was in 50 ml culture and culture volume could not be efficiently scaled up. 67 % pure VLP was produced with ultracentrifugation, while 100 % pure VLP was produced with IEX. Yield for 100 % pure VLP was 0.6 mg/l.
Conclusion: Highest VLP yield was achieved with High Five insect cells using 5 days baculovirus infection, 50 ml culture volume, flashBAC GOLD or ULTRA and MOI 1. 100 % pure CVB1-VLP was generated using three step IEX.
Methods: Baculovirus-insect cell expression system was used in the production of CVB1- VLPs. Various steps in the CVB1-VLP production were optimized including the type of insect cell line and the flashBAC DNA variant, the Multiplicity Of Infection (MOI) used for CVB1-VLP amplification, baculovirus cultivation time, type of cell growth medium and culture volumes and the CVB1-VLP purification method. VLPs were concentrated either using tangential flow filtration or PEG-precipitation and purified using multi-step ion exchange chromatography (IEX) purification. The quality and the purity of the CVB1- VLPs were estimated with SDS-PAGE, Western Blotting, dynamic light scattering and Transmission Electron Microscopy analyzes.
Results: The highest CVB1-VLP production level was obtained in High Five cells and they expressed two times more extracellular VLP than Sf9 cells. FlashBAC ULTRA and flashBAC GOLD expressed considerably more extracellular VLP than flashBAC or flashBAC PRIME in High Five cells. Further, the production levels of flashBAC ULTRA and flashBAC GOLD were almost equal. MOI 1 seemed to give the highest production levels. Highest yield was in 50 ml culture and culture volume could not be efficiently scaled up. 67 % pure VLP was produced with ultracentrifugation, while 100 % pure VLP was produced with IEX. Yield for 100 % pure VLP was 0.6 mg/l.
Conclusion: Highest VLP yield was achieved with High Five insect cells using 5 days baculovirus infection, 50 ml culture volume, flashBAC GOLD or ULTRA and MOI 1. 100 % pure CVB1-VLP was generated using three step IEX.