Production and Purification of Recombinant Hepatitis B Virus Particles

Abstract

Source: Nishitsuji, H., et al. Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle. J. Vis. Exp. (2017)

This video demonstrates the production and purification of recombinant Hepatitis B virus particles, enabling safe, replication-deficient viral preparations for studying early infection events and for high-throughput antiviral drug screening applications.

Protocol

1. Production of Recombinant HBV (Hepatitis B Virus) Encoding the Reporter Protein

1. Preparation of HepG2 cells
   1. Prepare cell culture medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml nonessential amino acids).
   2. Plate 4 x 10⁶ HepG2 (Human Hepatocellular Carcinoma G2) cells in a 10-cm collagen-coated dish in 10 ml of culture medium the day before transfection. Incubate HepG2 cells at 37 °C in a humidified 5% CO₂ incubator.
      NOTE: When approximately 4 x 10⁶ HepG2 cells are plated in a 10 cm collagen-coated dish in 10 ml of culture medium, they will be 70-90% confluent the next day.
2. Transfection
   1. Transfect HepG2 cells with 5 µg of pUC1.2HBV delta epsilon15 (an HBV replication plasmid with a deletion in the epsilon sequence) and 5 µg of pUC1.2HBV/NL15 using a transfection reagent as per manufacturer's instructions.
   2. The next day, remove the culture medium and add 10 ml of fresh culture medium.
   3. One week after transfection, transfer the culture medium containing the recombinant HBV to a 50 ml-tube and proceed to step 1.3.1. Add 10 ml of fresh culture medium to the plate containing the transfected cells.
      NOTE: Production of the recombinant HBV is maintained for 4 weeks. The culture medium containing recombinant HBV can be stored at 4 °C for 1 month.
3. Purification of recombinant HBV
   1. Remove cell debris from the culture medium containing recombinant HBV by centrifugation (2,300 x g for 5 min).
   2. Pass the supernatant through a 0.45 µm membrane filter.
   3. Add an equal volume of 26% PEG/1.5 M NaCl (polyethylene glycol 6000: 130 g, NaCl (sodium chloride), 49 g, 1 ml of 0.5 M EDTA (Ethylenediamine tetraacetic acid) pH 8.0 and 5 ml of 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) pH 7.6 in 500 ml) to the culture medium containing recombinant HBV and mix gently. Incubate overnight at 4 °C.
   4. Centrifuge at 2,300 x g for 20 min at 4 °C.
   5. Discard the supernatant and dissolve the pellet in 0.5 ml of TNE (10 mM Tris, 50 mM NaCl, 1 mM EDTA).
   6. Remove the debris by centrifugation at 2,300 x g for 5 min.
   7. Load 0.5 ml of TNE containing recombinant HBV onto 0.8 ml of 20% sucrose in TNE.
   8. Centrifuge at 100,000 × g for 3 hr at 15 °C.
   9. Discard as much of the supernatant as possible, and save the pellet. Resuspend it in 1 ml of serum free DMEM per 40 ml of starting culture medium.
   10. Incubate overnight at 4 °C.
   11. Filter through a 0.45 µm filter. Prepare 0.5 ml aliquots and store at -80 °C.
       NOTE: If reporter protein contamination of the original virus sample is observed, purify the virus by density gradient ultra-centrifugation of 5-30% sucrose in TNE at 100,000 x g for 2 hr, or CsCl (cesium chloride) density equilibrated centrifugation from 1.1-1.6 g/ml at 150,000 x g for 50 hr.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nano-Glo Luciferase Assay Regent	Promega	N1110
Penicillin-Streptomycin Mixed Solution	Nacalai tesque	09367-34
DMEM	Thermo Fisher Scientific	11995065
100 mm/collagen-coated dish	Iwaki	4020-010
Lipofectamine 3000 Transfection Reagent	Thermo Fisher Scientific	L3000001
Polyethylene glycol (PEG) 6000	Sigma-Aldrich	81255
Polyethylene glycol (PEG) 8000	Sigma-Aldrich	89510
NaCl	Nacalai tesque	31319-45
0.5 mol/L EDTA Solution	Nacalai tesque	06894-14
Tris-HCl	Nacalai tesque	35434-21
Millex-HP, 0.45 μm, polyethethersulfone, filter	Merck Millipore	SLHP033RS
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650
Collagen coated 96-well plate	Corning	NO3585
HepG2-NTCP1-myc-clone22	-	-
pUC1.2HBV delta epsilon	-	-
pUC1.2HBV/NL	-	-