Testing a novel method for protein - assembly based vaccine construction
Badal, Nasir Uddin (2019)
2019
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2019-06-28
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202202172006
https://urn.fi/URN:NBN:fi:tuni-202202172006
Tiivistelmä
Vaccination is thought to be the most effective and versatile component against infectious diseases. Emerging infectious diseases and limited effectiveness of currently available influenza vaccine can cause a serious public health threat to people worldwide. Millions of people are affected in yearly influenza outbreaks and more than 250,000 lives are lost annually. This study aimed to create a universal influenza vaccine prototype that will be cost effective, easy to produce, would provide long-term immunity and effectiveness against multiple strains of influenza and would be highly immunogenic. This kind of vaccine can be possibly formed by conjugating conserved M2e antigen and Norovirus-like particle (NoV-VLP) using two peptide tags (Spy-Tag and K-Tag) and Spyligase protein.
The extracellular domain of influenza A virus matrix protein-2 (M2e) was aimed to use as target antigen. M2e antigen was fused with a peptide tag (K-Tag) for the formation of isopeptide bond with another peptide tag (Spy-Tag) with the help of Spyligase. Spyligase is the essential enzyme to form covalent bond between K-Tag-Spy-Tag, that was expressed in BL21 Star DE3 E. coli strain. After complete purification of Spyligase using immobilized metal affinity chromatography (IMAC) method, 86 mg/l of Spyligase protein was obtained with ˃95% purity. Spy-Tag was genetically fused with Noro VLP so that, VLP could be decorated and conjugated with K-Tagged antigen. Spy Noro VLP was expressed in Hi-5 insect cells and purified with tangential flow filtration (TFF), 30 % sucrose cushion pelleting and anion exchange chromatography. Estimated yield from this purification was 133 mg/l with ˃95% purity. WT Noro VLP was purified using the same protocol as Spy Noro VLP and yield was 3.8 mg/l with ˃95% or ˃50% purity. Hydrodynamic diameter for Spy Noro VLP was 51 nm and for >95% pure WT Noro VLP and >50% pure WT Noro VLP were 53 nm and 32 nm respectively.
Spy-Tagged Noro virus like particle, K-Tagged antigen and Spyligase were mixed together and incubated at 4ºC for 24 hours. SDS-PAGE and western blot analysis showed the isopeptide bond formation and successful conjugation of two peptide tag.
The extracellular domain of influenza A virus matrix protein-2 (M2e) was aimed to use as target antigen. M2e antigen was fused with a peptide tag (K-Tag) for the formation of isopeptide bond with another peptide tag (Spy-Tag) with the help of Spyligase. Spyligase is the essential enzyme to form covalent bond between K-Tag-Spy-Tag, that was expressed in BL21 Star DE3 E. coli strain. After complete purification of Spyligase using immobilized metal affinity chromatography (IMAC) method, 86 mg/l of Spyligase protein was obtained with ˃95% purity. Spy-Tag was genetically fused with Noro VLP so that, VLP could be decorated and conjugated with K-Tagged antigen. Spy Noro VLP was expressed in Hi-5 insect cells and purified with tangential flow filtration (TFF), 30 % sucrose cushion pelleting and anion exchange chromatography. Estimated yield from this purification was 133 mg/l with ˃95% purity. WT Noro VLP was purified using the same protocol as Spy Noro VLP and yield was 3.8 mg/l with ˃95% or ˃50% purity. Hydrodynamic diameter for Spy Noro VLP was 51 nm and for >95% pure WT Noro VLP and >50% pure WT Noro VLP were 53 nm and 32 nm respectively.
Spy-Tagged Noro virus like particle, K-Tagged antigen and Spyligase were mixed together and incubated at 4ºC for 24 hours. SDS-PAGE and western blot analysis showed the isopeptide bond formation and successful conjugation of two peptide tag.