Generation of virosome particles
A virion, particle technology, applied in the direction of virus/phage, virus, antiviral agent, etc., can solve the problem of no provision, no suggestion, etc.
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Embodiment 1
[0174] Example 1: Preparation of Virosome Particles
[0175] Influenza hemagglutinin expressed and purified from N. benthamiana dissolved in PBS was mixed with powdered egg-derived lipid (lecithin such as egg yolk lecithin) dissolved in PBS containing 100 mM OEG as a detergent. The lipoprotein ratio can vary from 20:1 to 1:10. In our experiments, the optimal ratio was 6:1. If other lipids (synthetic or steroid types) are used, the lipid-protein ratio can vary even more. Lipid and influenza HA can optionally be subjected to pulses of ultrasound. The mixture was then passed through a 0.22mm filter and passed through a series of different channels in the SM-2 Bio-Bead to remove detergent. Removal of the detergent allows spontaneous assembly of the mixture of dissolved components in the virosome particle population. After the last pass of the SM-2 Bio-Bead, the virion particle population was again subjected to 0.22 mm filtration and the final product was a homogeneous populati...
Embodiment 2
[0176] Example 2: Alternative Modifications of Virosome Particle Production
[0177] Powdered lipid mixtures such as egg-derived phosphatidylcholine and phosphatidylethanolamine were dissolved in PBS containing 100 mM OEG as a detergent. Lipid ratios can vary from 20:1 to 1:10. In our example, the optimal range is 5:1. Lipid ratios can vary from 20:1 to 1:10. In our example, the optimum ratio is 7:1. If other lipids (synthetic or steroid types) are used, the lipid-protein ratio can vary even more. Lipid and influenza HA can optionally be subjected to pulses of ultrasound.
[0178] The solution was then mixed with influenza hemagglutinin expressed and purified from N. benthamiana in PBS. Lipid and influenza HA can optionally be subjected to pulses of ultrasound. The mixture was then passed through a 0.22mm filter and passed through a series of different channels in the SM-2 Bio-Bead to remove detergent. In the final step, the detergent was removed by batch chromatography...
Embodiment 3
[0180] Example 3: Sucrose gradient and silver staining
[0181]Sucrose gradient: By discontinuous ultracentrifugation, a sucrose gradient was used as an analytical method to assess antigen binding in virion particles based on the different densities of individual components. Aliquots of virosome particle preparations in PBS were applied on top of a 10-60% (w / v) discontinuous sucrose gradient in PBS and centrifuged at 100,000 g for 24 h at 4°C. The collected fractions were then analyzed for density and followed by SDS PAGE and silver staining to identify HA-containing fractions.
[0182] Silver staining: SDS PAGE was performed according to the manufacturer's instructions (Invitrogen). Silver staining was performed according to the manufacturer's instructions (Bio-Rad).
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