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 collagen coated dish. Incubate the cells at 37 degrees Celsius in a humidified 5%CO₂ incubator. Using a transfection agent according to the manufacturer's instructions, transfect the cells with 5 mcg of pUC1.2HBVΔε, and 5 mcg of pUC1.2HBV/NL.

The next day, remove the culture medium and add 10 mL of fresh culture medium. Then, one week after transfection, transfer the culture medium containing the recombinant HBV to a 50 mL tube.

Then collect and centrifuge the culture medium at 2,300 x g for five minutes to remove the cell debris containing the recombinant HBV. Pass the supernatant through a 0.45 mcm membrane filter. Then add an equal volume of 25%PEG, 1.5 molar NaCl, to the filtrate, and gently mix.

Incubate the sample overnight at four degrees Celsius. The following day, spin the solution at 2,300 x g and four degrees Celsius, for 20 minutes. Discard the supernatant and dissolve the pellet in 0.5 mL of TNE buffer. then load 0.5 mL of the TNE-containing recombinant HBV onto 0.8 mL of 20%sucrose in TNE.

Centrifuge the two at 100,000 x g and 15 degrees Celsius for three hours. Discard as much of the supernatant as possible and use 1 mL of serum-free DMEM per 40 mL of starting culture to re-suspend the pellet. Incubate the suspension at four degrees Celsius overnight. The following morning, filter the suspension through a 0.45 mcm filter. Then prepare 0.5 mL aliquots and store the tubes at minus 80 degrees Celsius.