Abstract
Antiviral agents for the suppression of hepatitis B virus (HBV) have been used for treating chronic hepatitis B. However, the emergence of drug-resistant HBV is still a major problem for antiviral treatment. To identify and characterize the drug-resistant HBV, the construction of HBV replicon and in vitro drug susceptibility assay are essential. Here we describe the experimental methods to study drug-resistant HBV.
Keywords: Hepatitis B virus, Antiviral agent, Drug susceptibility, Drug resistant mutation, Southern blot
Materials and Reagents
-
Transfection HBV 1.2mer replicons
-
Huh7 cells (Liver Research Center, Rhode Island Hospital, Brown Medical School, Providence, USA)
-
Dulbecco modified Eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 11995 )
-
Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 26140-079 )
-
Penicillin-streptomycin (Life Technologies, Gibco®, catalog number: 15140 )
-
Lipofectamine 2000 (Life Technologies, InvitrogenTM, catalog number: 11668-019 )
-
Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Life Technologies, Gibco®, catalog number: 31985-088 )
-
HBV 1.2mer replicon [WT or Drug-resistant HBV, WT HBV1.2 replicon
was kindly provided from Prof. W. S. Ryu (Yonsei University, Korea)]
-
Lamivudine (1~100 μM, provided by GlaxoSmithKline)
-
Adefovir (1~100 μM, provided by Gilead Sciences)
-
Clevudine (1~100 μM, provided by Bukwang Pharmaceutical Co.)
-
Entecavir (0.05~1 μM, Moravek)
-
Tenofovir (1~100 μM, provided by Gilead Sciences)
-
Extraction of viral DNA from HBV core particles
-
DNaseI (Takara Bio Company, catalog number: 2215B )
-
MungBean Nuclease (Takara Bio Company, catalog number: 2420B )
-
Proteinase K (Roche Diagnostics, catalog number: 03115801001 )
-
Phenol-chloroform-isoamyl alcohol 25:24:1 (Sigma-Aldrich, catalog number: 77617 )
-
Sodium acetate trihydrate (NaOAc) (Sigma-Aldrich, catalog number: 236500 )
-
HEPES lysis buffer (see Recipes)
-
Nuclease buffer (I) (see Recipes)
-
26% PEG solution (see Recipes)
-
Nuclease buffer (II) (see Recipes)
-
0.5% SDS solution (see Recipes)
-
Southern blot assay and the construction of the HBV probe
-
Certified Molecular Biology Agarose (Bio-Rad Laboratories, catalog number: 161-3102 )
-
Amersham Hybond-N+ membrane (GE Healthcare, catalog number: RPN203B )
-
SSC Buffer (Sigma-Aldrich, catalog number: 85639 )
-
Salmon sperm DNA (Life Technologies, InvitrogenTM, catalog number: AM9680 )
-
Poly ethylene glycol (PEG) (Sigma-Aldrich, catalog number: P5413 )
-
50x Denhardt’s solution (Sigma-Aldrich, catalog number: D2532 )
-
0.1 M sodium phosphate buffer (pH 7.0)
-
Formamide (Sigma-Aldrich, catalog number: F7503 )
-
HBV 1.2mer replicon
-
Klenow fragment and 10x Klenow buffer (Takara Bio Company, catalog number: 2140B )
-
dNTP (NEB, catalog number: N0446S )
-
[α-32P] dCTP (3,000 Ci/mmole, Perkin Elmer, catalog number: BLU513H500UC )
-
Quick Spin Columns for radiolabeled DNA purification (Sephadex G-50, Roche Diagnostics, catalog number: 11 273 973 001 )
-
Aat II (New England BioLabs, catalog number: R0117S )
-
Hind III (New England BioLabs, catalog number: R0104S )
-
Random primer (Bioneer, catalog number: N-7052 )
-
Na2HPO4 (Amresco, catalog number: 0404 )
-
NaH2PO4 (Amresco, catalog number: 0571 )
-
0.1 M sodium phosphate buffer (see Recipes)
-
Transfer solution (I) (see Recipes)
-
Transfer solution (II) (see Recipes)
-
Transfer solution (III) (see Recipes)
-
Hybridization solution (see Recipes)
-
Washing solution (I) (see Recipes)
-
Washing solution (II) (see Recipes)
Equipment
-
37 °C, 5% CO2 forced-air incubator (Thermo Fisher Scientific, Forma®, model: 3131 )
-
100 mm cell culture dish (Nunc®, catalog number: 172958 )
-
6 well plate (Nunc®, catalog number: 140675 )
-
Centrifuge (Eppendorf, catalog number: 5415R )
-
Pipet Aid XP (Drummond Scientific Company)
-
Electrophoresis system (Nihon Eido, catalog number: NB-1011 )
-
Dry oven
-
Hybridization bottle
-
Hybridization chamber
-
Heat block
Procedure
-
Transfection of HBV 1.2mer replicons
-
One day before transfection, plate 3 x 105 Huh7 cells per well in 2 ml
of growth medium (DMEM with 10% FBS, 1% Penicillin-streptomycin) on 6
well plate.
-
Two hours before transfection, change 2 ml of growth media per well at 80% cells confluence.
-
Dilute the 2 μg of HBV 1.2mer replicons in 125 μl of Opti-MEM and gently flick.
-
Dilute 4 μl of Lipofectamine 2000 in 125 μl of Opti-MEM and gently flick. Then, incubate for 5 min at room temperature.
-
After 5 min incubation, combine the diluted HBV 1.2mer replicons
with the diluted Lipofectamine 2000 and gently mix by pipetting. Then,
incubate for 20 min at room temperature.
-
After 4~6 h
transfection, change 2 ml of growth medium per well mixed with
appropriate concentration anti-HBV drugs (see Figure 2).
-
Replace
the growth medium with anti-HBV drugs every day for 4 days and, then
harvest the cells in 1.5 ml tube by centrifugation at 3,000 rpm for 1
min at 4 °C.
-
Extraction of core particle relative HBV DNA
-
Add 100 μl of cold HEPES lysis buffer in cell pellet and incubate on ice for 20 min.
-
Centrifuge the lysates at 13,000 rpm for 10 min at 4 °C and transfer
supernatant (cell lysate, cytoplasm fraction) into new 1.5 ml tube.
-
Mix 5.67 μl of nuclease buffer (I) by pipetting and incubate for 30 min in 37 °C water bath.
-
Briefly spin down, add 40 μl of 26% PEG solution and incubate for 2 h
on ice (vortexing every 30 min) and centrifuge at 13,000 rpm for 30 min
at 4 °C.
-
Discard the supernatant and centrifuge at 13,000 rpm for 5 min at 4 °C.
-
Completely remove the supernatant (using pipet and KIMTECH Science Wipers).
-
Add 100 μl of nuclease buffer (II) and physically resuspend the core particles.
-
Incubate for 20 min in 37 °C water bath and briefly spin down.
-
Add 282.5 μl of 0.5% SDS solution and 12.5 μl of proteinase K (20
mg/ml) and incubate for 2~4 h in 37 °C water bath (inverting every 30
min).
-
After spin down, add 400 μl of phenol-chloroform-isoamyl
alcohol and vigorously vortex for 10 sec. Then, centrifuge at 13,000 rpm
for 5 min at room temperature.
-
Transfer aqueous upper layer into new 1.5 ml tube.
-
Add 40 μl of 3 M NaOAc and 800 μl of 100% ethanol. After inverting
several times, precipitate the mixture overnight at -20 °C.
-
Centrifuge at 13,000 rpm for 30 min at 4 °C and throw away the supernatant.
-
Add 800 μl of 70% ethanol and gently inverting.
-
Centrifuge at 13,000 rpm for 10 min at 4 °C and throw away the supernatant.
-
Add 800 μl of 100% ethanol and gently inverting.
-
Centrifuge at 13,000 rpm for 10 min at 4 °C and throw away the supernatant.
-
Dry the HBV DNA pellet for 5~10 min (air-drying) and dissolve in 15 μl of TE buffer. Store HBV DNA at 4 °C until use.
-
Southern blot assay (gel transfer)
-
Separate HBV DNA (procedure B, step 18) on 0.8% agarose gel for 2 h at 89 V.
-
After electrophoresis, transfer the gel to tray and add transfer solution (I) to soak the gel.
-
Incubate for 10 min on shaker and rinse the gel with distilled water.
-
Add transfer solution (II) and incubate for 45 min on shaker.
-
Rinse with distilled water and add transfer solution (III).
-
Incubate over 30 min on shaker and rinse the gel with distilled water.
-
Transfer the gel to Hybond-N+ membrane for overnight using capillary
method (Figure 1) and bake the membrane to fix the HBV DNA for 2 h in
80 °C dry oven.
-
Place membrane in hybridization bottle contained
with 25 ml hybridization solution and incubates the membrane for 4 h in
42 °C hybridization chamber (pre-hybridization step).
-
The construction of the HBV probe
-
To construct a template for the HBV specific probe, digest HBV 1.2mer
replicon with Aat II and Hind III for 2 h at 37 °C and gel purification
(Ahn et al., 2015).
-
To synthesis HBV specific probe, mix the 48 ng of template (step 8) and 80 ng of random primer in 50 μl of distilled water.
Note: Step 10 should be carried out during pre-hybridization step.
-
Heat the probe mixtures for 5 min at 100 °C and snap cooling on ice for 5 min.
-
After spin down, add 5ul of 10x Klenow buffer, 2.5 μl of the dNTPs
mixtures (2.5 mM, except dCTP), 5 μl of 32P-labled dCTP (50 μCi=5
μl/labeling), and 1 μl of Klenow fragment in probe mixtures.
-
Incubate the probe mixture for 30 min at 37 °C.
-
Remove the non-labeled probe using Quick Spin Columns for radiolabeled DNA purification.
-
Southern blot assay (membrane hybridization)
-
Replace pre-warmed 25 ml of hybridization solution with 32P-labled HBV
probe and incubate overnight in 42 °C hybridization chamber.
-
After hybridization step, wash the membrane with washing solution (I) for 15~20 min in 63 °C hybridization chamber.
Note: If need more washing step, repeat wash with washing solution
(II). Steps 9~16 should be performed in the Radio isotope room.
-
Expose membrane to film and incubate overnight at -80 °C deep freezer. Then, develop the film.
Representative data

Figure 1. Scheme of capillary transfer method for Southern blotting

Figure 2. In vitro susceptibility assay of drug-resistant HBV (Kim et al., 2014). The replication of drug-resistant HBV was analyzed by Southern blot. TDF and ETV were treated with concentration as followed table (below).
Recipes
-
HEPES lysis buffer
10 mM Hepes (pH 7.5)
100 mM NaCl
1 mM EDTA
1% NP-40
-
Nuclease buffer (I)
10 mM CaCl2
12 mM MgCl2
DNaseI 0.5 unit
Mung Bean nuclease 7.5 unit
-
26% PEG solution
1.2 M NaCl
60 mM EDTA
30% sucrose
26% PEG 8000
-
Nuclease buffer (II)
10 mM Tris (pH 7.5)
8 mM CaCl2
6 mM MgCl2
DNaseI 2 unit
Mung Bean nuclease 3 unit
-
0.5% SDS solution
25 mM Tris (pH 7.5)
10 mM EDTA
100 mM NaCl
0.5% SDS
-
0.1 M sodium phosphate buffer (pH 7.0)
57.7 ml of 1 M Na2HPO4
42.3 ml of 1 M NaH2PO4
-
Transfer solution (I)
0.25 N HCl
0.6 M NaCl
-
Transfer solution (II)
0.5 M NaOH
1 M NaCl
-
Transfer solution (III)
1 M Tris (pH 7.5~8.0)
1 M NaCl
-
Hybridization solution
Formamide 25 ml
20x SSC 12.5 ml
0.1 M sodium phosphate buufer (pH 7.0) 2.5 ml
0.5 M EDTA 0.2 ml
50x Denhardt’s solution 3 ml
10% SDS 1 ml
Salmon sperm DNA (10 mg/ml) 1 ml
Distilled water 4.8 ml
-
Washing solution (I)
2x SSC
0.1% SDS
-
Washing solution (II)
0.5x SSC
0.1% SDS
Acknowledgments
This study was supported by Konkuk University.
References
-
Ahn, S. H., Park, Y. K., Park, E. S., Kim, J. H., Kim, D. H., Lim, K. H., Jang, M. S., Choe, W. H., Ko, S. Y., Sung, I. K., Kwon, S. Y. and Kim, K. H. (2014). The impact of the hepatitis B virus polymerase rtA181T mutation on replication and drug resistance is potentially affected by overlapping changes in surface gene. J Virol 88(12): 6805-6818.
-
Ahn, S. H., Park, Y. K. and Kim, K. (2015). Introduction and sequencing of patient-isolated HBV RT sequences into the HBV 1.2-mer replicon. Bio-protocol 5(8): e1449.
-
Kim, J. H., Park, Y. K., Park, E. S. and Kim, K. H. (2014). Molecular diagnosis and treatment of drug-resistant hepatitis B virus. World J Gastroenterol 20(19): 5708-5720.
-
Kwon, S. Y., Park, Y. K., Ahn, S. H., Cho, E. S., Choe, W. H., Lee, C. H., Kim, B. K., Ko, S. Y., Choi, H. S., Park, E. S., Shin, G. C. and Kim, K. H. (2010). Identification and characterization of clevudine-resistant mutants of hepatitis B virus isolated from chronic hepatitis B patients. J Virol 84(9): 4494-4503.
Please login or register for free to view full text
View full text
Download PDF
Q&A
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Ahn, S. H., Park, Y. K. and Kim, K. (2015).
In vitro Drug Susceptibility Assay for HBV Using Southern Blotting.
Bio-protocol 5(8): e1448. DOI:
10.21769/BioProtoc.1448.