Generation of Chikungunya Virus-Like Particles Using Baculovirus Expression System in Insect Cells

Overview

This video shows how virus-like particles, VLPs are generated by infecting insect cells with a recombinant baculovirus carrying the chikungunya virus gene cassette. The recombinant virus infects cells, leading to VLP formation, assembly, and release into the culture medium for potential use in vaccine production.

Protocol

1. Chikungunya virus-like particles (VLP) Expression Using Baculovirus/Insect Cell System

1. Generate recombinant baculovirus expressing chikungunya virus capsid and envelope proteins (C-E3-E2-6K-E1) from S-27 strain using a commercial baculovirus expression system.
2. Culture Spodoptera frugiperda Sf9 cells in serum-free Sf9 growth medium with 100 U/ml penicillin and 100 µg/ml streptomycin at 28 °C. Use 3-5 x 10⁵ c/ml to initiate spinner flask cultures for suspension cell growth. Passage routinely when they reach cell densities of 2-4 x 10⁶ c/ml (every 3-4 days).
3. Culture Sf9 cells in suspension in spinner flasks with stirring at 130 rpm on a multipoint stirrer plate system. For proper aeration, maintain culture volumes at no more than half the volume of the spinner flask.
4. For expression of Chikungunya or CHIK VLPs, infect Sf9 cells at a density of 2 x 10⁶ cells/ml with recombinant baculovirus at a multiplicity of infection of 1 and return to 28 °C incubator. Typically, infect one or two spinner flasks, containing 250 ml Sf9 cells.
   Note: While we do not use this method, Sf9 cells may be cultured in shaker flasks at 28 °C using a shaker platform.
5. Harvest cultures after 72-96 hours post-infection, or when cell viability has decreased to 70-80% as determined by Trypan Blue Exclusion according to the manufacturer's protocol.
   Note: The cells continue to proliferate while infected and there is a ~80% viability in Sf9 cultures infected with recombinant CHIK VLP baculovirus at 3 days post-infection (dpi). Baculovirus-infected cells are larger in appearance. Morphology may also change from round to oblong. In the late stages of baculovirus infections, the cells began to lyse.
6. Transfer cultures directly from suspension culture into 50 ml conical tubes and spin the cells down at 500 x g for 5 min at 4 °C.
7. Collect supernatants and filter through a 0.22 µm pore membrane before sedimentation via ultracentrifugation.

Materials

Name	Company	Catalog Number	Comments
Opti-MEM I	Life Technologies	31985062
Bac-to-Bac Baculovirus Expression System	Life Technologies	10359-016
0.22 μm vacuum filter top (500 mL)	Nalgene	569-0020
Sf-900 II SFM	ThermoFisher Scientific	10902-096
Cimarec I 6 multipoint stirrer plate	ThermoFisher Scientific	50094596