A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Samenvatting

Source: Arevalo, M. T., et al.  Expression and Purification of Virus-like Particles for Vaccination. J. Vis. Exp. (2016).

This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors — encoding different viral structural proteins — in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

Protocol

1. Mammalian Expression System for Generation of Influenza H3N2 VLP

1. Subclone viral structural glycoproteins hemagglutinin (HA), neuraminidase (NA), and human immunodeficiency virus (HIV) Gag (group-specific antigen) p55 into eukaryotic expression vectors, such as previously described pTR600.
2. Amplify DNA in chemically competent Escherichia coli (e.g., DH5α) and isolate transfection-grade plasmid using a plasmid purification kit per manufacturer's instructions. The amount of DNA is dependent on yield and the user's needs.
3. Maintain mammalian cell line, 293T, in growth media containing 10% fetal bovin....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Plasmid Maxi Kit	Qiagen	12163
DMEM	Cellgro	10-013
Lipofectamine	Life Technologies	L3000075	Lipofectamine 2000, Lipofectamine 3000
Opti-MEM I	Life Technologies	31985062
Cimarec I 6 multipoint stirrer plate	ThermoFisher Scientific	50094596
Polyclear ultracentrifuge tubes	Seton	7025
0.22 μm vacuum filter top (500 mL)	Nalgene	569-0020
Glycerol	Sigma	G5516