An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection
Published: October 31, 2023
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 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.
Protocol
1. Production of Recombinant HBV Encoding the Reporter Protein Preparation of HepG2 cells Prepare cell culture medium (Dulbecco's modified Eagle's medium or DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml nonessential amino acids). Plate 4 x 106 HepG2 cells in a 10-cm collagen-coated dish in 10 ml of culture medium the day before transfection. Incubate HepG2 cells at…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Tags
Cite This Article
An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection. J. Vis. Exp. (Pending Publication), e21756, doi: (2023).