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 an in vitro technique to detect hepatitis B virus (HBV) infection via a recombinant HBV reporter system. Upon infecting host cells, the recombinant viral genome expresses the luciferase reporter enzyme, which produces bioluminescence upon oxidizing its substrate — helping detect viral infection.
1. Production of Recombinant HBV Encoding the Reporter Protein
2. Infection of Recombinant HBV
3. Analysis
The authors have nothing to disclose.
Nano-Glo Luciferase Assay Regent | Promega | N1110 | |
Penicillin-Streptomycin Mixed Solution | Nacalai tesque | 09367-34 | |
MEM Non-Essential Amino Acids Solution | Thermo Fisher Scientific | 11140050 | |
DMEM | Thermo Fisher Scientific | 11995065 | |
Opti-MEM I Reduced Serum Medium | Thermo Fisher Scientific | 31985070 | |
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 | |
Passive Lysis 5xBuffer | Promega | E1941 | |
GloMax 96 Microplate Luminometer | Promega | E6501 | |
Sucrose | Nacalai tesque | 30403-55 | |
Luminometer plate | Greiner bio-one | 655075 | |
HepG2-NTCP1-myc-clone22 | – | – | Nishitsuji, H., et al. Cancer. Sci. (2015) |
pUC1.2HBV delta epsilon | – | – | Nishitsuji, H., et al. Cancer. Sci. (2015) |
pUC1.2HBV/NL | – | – | Nishitsuji, H., et al. Cancer. Sci. (2015) |
50 ml tube | Violamo | 1-3500-02 | |
Anti-Myc antibody | Sigma-Aldrich | C3956 | |
HBIG | Japan Blood Products Organization | – | |
IFN-β | Mochida Pharmaceutical | 1.49872E+13 | |
Heparin | Sigma-Aldrich | H3393 |