Production and Purification of Recombinant Hepatitis B Virus Particles

Begin with hepatocytes cultured on a collagen-coated dish.

Introduce a transfection reagent complexed with two plasmids: one carrying a modified Hepatitis B Virus or HBV genome, and the other encoding the viral structural proteins.

Incubate to allow plasmid entry. Inside the cell, viral genes are expressed to produce structural proteins and a modified HBV genome, which assemble to form recombinant HBV particles.

Replace the medium and incubate to support viral particle production and release.

Harvest the medium and centrifuge to pellet the debris.

Collect the supernatant and filter it to remove remaining debris.

Next, add polyethylene glycol and sodium chloride to precipitate viral particles.

Centrifuge to pellet the contents. Dissolve the pellet in buffer and spin down the debris. Then, layer it onto a sucrose cushion.

Perform ultracentrifugation to sediment viral particles through the cushion.

Remove the supernatant and resuspend the pellet in medium.

Filter the suspension and store the purified recombinant HBV at low temperature for downstream analysis.

After preparing cell culture medium, the day before transfection, place four times 10 to the six HepG2 cells in medium, on a 10 cm collage