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In vitro Drug Susceptibility Assay for HBV Using Southern Blotting
采用Southern蛋白印迹法进行HBV体外药敏性试验   

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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(乙型肝炎病毒), Antiviral agent(抗病毒药物), Drug susceptibility(药物敏感性), Drug resistant mutation(耐药突变), Southern blot(Southern印迹杂交)

Materials and Reagents

  1. Transfection HBV 1.2mer replicons
    1. Huh7 cells (Liver Research Center, Rhode Island Hospital, Brown Medical School, Providence, USA)
    2. Dulbecco modified Eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 11995 )
    3. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 26140-079 )
    4. Penicillin-streptomycin (Life Technologies, Gibco®, catalog number: 15140 )
    5. Lipofectamine 2000 (Life Technologies, InvitrogenTM, catalog number: 11668-019 )
    6. Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Life Technologies, Gibco®, catalog number: 31985-088 )
    7. HBV 1.2mer replicon [WT or Drug-resistant HBV, WT HBV1.2 replicon was kindly provided from Prof. W. S. Ryu (Yonsei University, Korea)]
    8. Lamivudine (1~100 μM, provided by GlaxoSmithKline)
    9. Adefovir (1~100 μM, provided by Gilead Sciences)
    10. Clevudine (1~100 μM, provided by Bukwang Pharmaceutical Co.)
    11. Entecavir (0.05~1 μM, Moravek)
    12. Tenofovir (1~100 μM, provided by Gilead Sciences)

  2. Extraction of viral DNA from HBV core particles
    1. DNaseI (Takara Bio Company, catalog number: 2215B )
    2. MungBean Nuclease (Takara Bio Company, catalog number: 2420B )
    3. Proteinase K (Roche Diagnostics, catalog number: 03115801001 )
    4. Phenol-chloroform-isoamyl alcohol 25:24:1 (Sigma-Aldrich, catalog number: 77617 )
    5. Sodium acetate trihydrate (NaOAc) (Sigma-Aldrich, catalog number: 236500 )
    6. HEPES lysis buffer (see Recipes)
    7. Nuclease buffer (I) (see Recipes)
    8. 26% PEG solution (see Recipes)
    9. Nuclease buffer (II) (see Recipes)
    10. 0.5% SDS solution (see Recipes)

  3. Southern blot assay and the construction of the HBV probe
    1. Certified Molecular Biology Agarose (Bio-Rad Laboratories, catalog number: 161-3102 )
    2. Amersham Hybond-N+ membrane (GE Healthcare, catalog number: RPN203B )
    3. SSC Buffer (Sigma-Aldrich, catalog number: 85639 )
    4. Salmon sperm DNA (Life Technologies, InvitrogenTM, catalog number: AM9680 )
    5. Poly ethylene glycol (PEG) (Sigma-Aldrich, catalog number: P5413 )
    6. 50x Denhardt’s solution (Sigma-Aldrich, catalog number: D2532 )
    7. 0.1 M sodium phosphate buffer (pH 7.0)
    8. Formamide (Sigma-Aldrich, catalog number: F7503 )
    9. HBV 1.2mer replicon
    10. Klenow fragment and 10x Klenow buffer (Takara Bio Company, catalog number: 2140B )
    11. dNTP (NEB, catalog number: N0446S )
    12. [α-32P] dCTP (3,000 Ci/mmole, Perkin Elmer, catalog number: BLU513H500UC )
    13. Quick Spin Columns for radiolabeled DNA purification (Sephadex G-50, Roche Diagnostics, catalog number: 11 273 973 001 )
    14. Aat II (New England BioLabs, catalog number: R0117S )
    15. Hind III (New England BioLabs, catalog number: R0104S )
    16. Random primer (Bioneer, catalog number: N-7052 )
    17. Na2HPO4 (Amresco, catalog number: 0404 )
    18. NaH2PO4 (Amresco, catalog number: 0571 )
    19. 0.1 M sodium phosphate buffer (see Recipes)
    20. Transfer solution (I) (see Recipes)
    21. Transfer solution (II) (see Recipes)
    22. Transfer solution (III) (see Recipes)
    23. Hybridization solution (see Recipes)
    24. Washing solution (I) (see Recipes)
    25. Washing solution (II) (see Recipes)

Equipment

  1. 37 °C, 5% CO2 forced-air incubator (Thermo Fisher Scientific, Forma®, model: 3131 )
  2. 100 mm cell culture dish (Nunc®, catalog number: 172958 )
  3. 6 well plate (Nunc®, catalog number: 140675 )
  4. Centrifuge (Eppendorf, catalog number: 5415R )
  5. Pipet Aid XP (Drummond Scientific Company)
  6. Electrophoresis system (Nihon Eido, catalog number: NB-1011 )
  7. Dry oven
  8. Hybridization bottle
  9. Hybridization chamber
  10. Heat block

Procedure

  1. Transfection of HBV 1.2mer replicons
    1. 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.
    2. Two hours before transfection, change 2 ml of growth media per well at 80% cells confluence.
    3. Dilute the 2 μg of HBV 1.2mer replicons in 125 μl of Opti-MEM and gently flick.
    4. Dilute 4 μl of Lipofectamine 2000 in 125 μl of Opti-MEM and gently flick. Then, incubate for 5 min at room temperature.
    5. 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.
    6. After 4~6 h transfection, change 2 ml of growth medium per well mixed with appropriate concentration anti-HBV drugs (see Figure 2).
    7. 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.

  2. Extraction of core particle relative HBV DNA
    1. Add 100 μl of cold HEPES lysis buffer in cell pellet and incubate on ice for 20 min.
    2. 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.
    3. Mix 5.67 μl of nuclease buffer (I) by pipetting and incubate for 30 min in 37 °C water bath.
    4. 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.
    5. Discard the supernatant and centrifuge at 13,000 rpm for 5 min at 4 °C.
    6. Completely remove the supernatant (using pipet and KIMTECH Science Wipers).
    7. Add 100 μl of nuclease buffer (II) and physically resuspend the core particles.
    8. Incubate for 20 min in 37 °C water bath and briefly spin down.
    9. 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).
    10. 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.
    11. Transfer aqueous upper layer into new 1.5 ml tube.
    12. Add 40 μl of 3 M NaOAc and 800 μl of 100% ethanol. After inverting several times, precipitate the mixture overnight at -20 °C.
    13. Centrifuge at 13,000 rpm for 30 min at 4 °C and throw away the supernatant.
    14. Add 800 μl of 70% ethanol and gently inverting.
    15. Centrifuge at 13,000 rpm for 10 min at 4 °C and throw away the supernatant.
    16. Add 800 μl of 100% ethanol and gently inverting.
    17. Centrifuge at 13,000 rpm for 10 min at 4 °C and throw away the supernatant.
    18. 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.

  3. Southern blot assay (gel transfer)
    1. Separate HBV DNA (procedure B, step 18) on 0.8% agarose gel for 2 h at 89 V.
    2. After electrophoresis, transfer the gel to tray and add transfer solution (I) to soak the gel.
    3. Incubate for 10 min on shaker and rinse the gel with distilled water.
    4. Add transfer solution (II) and incubate for 45 min on shaker.
    5. Rinse with distilled water and add transfer solution (III).
    6. Incubate over 30 min on shaker and rinse the gel with distilled water.
    7. 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.
    8. 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).

  4. The construction of the HBV probe
    1. 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).
    2. 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.
    3. Heat the probe mixtures for 5 min at 100 °C and snap cooling on ice for 5 min.
    4. 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.
    5. Incubate the probe mixture for 30 min at 37 °C.
    6. Remove the non-labeled probe using Quick Spin Columns for radiolabeled DNA purification.

  5. Southern blot assay (membrane hybridization)
    1. Replace pre-warmed 25 ml of hybridization solution with 32P-labled HBV probe and incubate overnight in 42 °C hybridization chamber.
    2. 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.
    3. 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

  1. HEPES lysis buffer
    10 mM Hepes (pH 7.5)
    100 mM NaCl
    1 mM EDTA
    1% NP-40
  2. Nuclease buffer (I)
    10 mM CaCl2
    12 mM MgCl2
    DNaseI 0.5 unit
    Mung Bean nuclease 7.5 unit
  3. 26% PEG solution
    1.2 M NaCl
    60 mM EDTA
    30% sucrose
    26% PEG 8000
  4. Nuclease buffer (II)
    10 mM Tris (pH 7.5)
    8 mM CaCl2
    6 mM MgCl2
    DNaseI 2 unit
    Mung Bean nuclease 3 unit
  5. 0.5% SDS solution
    25 mM Tris (pH 7.5)
    10 mM EDTA
    100 mM NaCl
    0.5% SDS
  6. 0.1 M sodium phosphate buffer (pH 7.0)
    57.7 ml of 1 M Na2HPO4
    42.3 ml of 1 M NaH2PO4
  7. Transfer solution (I)
    0.25 N HCl
    0.6 M NaCl
  8. Transfer solution (II)
    0.5 M NaOH
    1 M NaCl
  9. Transfer solution (III)
    1 M Tris (pH 7.5~8.0)
    1 M NaCl
  10. 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
  11. Washing solution (I)
    2x SSC
    0.1% SDS
  12. Washing solution (II)
    0.5x SSC
    0.1% SDS

Acknowledgments

This study was supported by Konkuk University.

References

  1. 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.
  2. 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.
  3. 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.
  4. 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.

简介

用于抑制乙型肝炎病毒(HBV)的抗病毒剂已经用于治疗慢性乙型肝炎。然而,耐药性HBV的出现仍然是抗病毒治疗的主要问题。 为了鉴定和表征耐药性HBV,构建HBV复制子和体外药物敏感性测定是必需的。 在这里我们描述研究耐药性HBV的实验方法。

关键字:乙型肝炎病毒, 抗病毒药物, 药物敏感性, 耐药突变, Southern印迹杂交

材料和试剂

  1. 转染HBV 1.2mer复制子
    1. Huh7细胞(肝脏研究中心,罗得岛医院,布朗医学院,普罗维登斯,美国)
    2. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:11995)
    3. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:26140-079)
    4. 青霉素 - 链霉素(Life Technologies,Gibco ,目录号:15140)
    5. Lipofectamine 2000(Life Technologies,Invitrogen TM ,目录号:11668-019)
    6. Opti-MEM I Reduced Serum Medium(Opti-MEM)(Life Technologies,Gibco ,目录号:31985-088)
    7. HBV 1.2mer复制子[WT或耐药HBV,WT HBV1.2复制子 由韩国延世大学教授W. S. Ryu提供]
    8. 拉米夫定(1〜100μM,由GlaxoSmithKline提供)
    9. 阿德福韦(1〜100μM,由Gilead Sciences提供)
    10. Clevudine(1〜100μM,Bukwang Pharmaceutical Co.提供)
    11. 恩替卡韦(0.05〜1μM,Moravek)
    12. 替诺福韦(1〜100μM,由Gilead Sciences提供)

  2. 从HBV核心颗粒提取病毒DNA
    1. DNaseI(Takara Bio公司,目录号:2215B)
    2. MungBean核酸酶(Takara Bio公司,目录号:2420B)
    3. 蛋白酶K(Roche Diagnostics,目录号:03115801001)
    4. 苯酚 - 氯仿 - 异戊醇25:24:1(Sigma-Aldrich,目录号:77617)
    5. 醋酸钠三水合物(NaOAc)(Sigma-Aldrich,目录号:236500)
    6. HEPES裂解缓冲液(见配方)
    7. 核酸酶缓冲液(I)(参见配方)
    8. 26%PEG溶液(参见配方)
    9. 核酸酶缓冲液(II)(参见配方)
    10. 0.5%SDS溶液(见配方)

  3. Southern印迹测定和HBV探针的构建
    1. 认证分子生物学琼脂糖(Bio-Rad Laboratories,目录号:161-3102)
    2. Amersham Hybond-N +膜(GE Healthcare,目录号:RPN203B)
    3. SSC缓冲液(Sigma-Aldrich,目录号:85639)
    4. 鲑鱼精子DNA(Life Technologies,Invitrogen TM,目录号:AM9680)
    5. 聚乙二醇(PEG)(Sigma-Aldrich,目录号:P5413)
    6. 50x Denhardt's溶液(Sigma-Aldrich,目录号:D2532)
    7. 0.1M磷酸钠缓冲液(pH7.0)
    8. 甲酰胺(Sigma-Aldrich,目录号:F7503)
    9. HBV 1.2mer复制子
    10. Klenow片段和10×Klenow缓冲液(Takara Bio Company,目录号:2140B)
    11. dNTP(NEB,目录号:N0446S)
    12. [α-32 P] dCTP(3000Ci/mmole,Perkin Elmer,目录号:BLU513H500UC)
    13. 用于放射性标记DNA纯化的快速离心柱(Sephadex G-50,Roche Diagnostics,目录号:11 273 973 001)
    14. Aat II (New England BioLabs,目录号:R0117S)
    15. Hind III (New England BioLabs,目录号:R0104S)
    16. 随机引物(Bioneer,目录号:N-7052)

    17. (Amresco,目录号:0404)。
    18. NaH 2 PO 4(Amresco,目录号:0571)
    19. 0.1 M磷酸钠缓冲液(见配方)
    20. 转移溶液(I)(参见配方)
    21. 转移溶液(II)(参见配方)
    22. 转移溶液(III)(参见配方)
    23. 100mm细胞培养皿(Nunc ,目录号:172958)
    24. 6孔板(Nunc ,目录号:140675)
    25. 离心机(Eppendorf,目录号:5415R)
    26. Pipet Aid XP(Drummond Scientific Company)
    27. 电泳系统(Nihon Eido,目录号:NB-1011)
    28. 干燥炉
    29. 杂交瓶
    30. 杂交室
    31. 热块

    程序

    1. HBV 1.2mer复制子的转染
      1. 转染前一天,将每孔3×10 5个Huh7细胞平板接种于2ml 的生长培养基(具有10%FBS,1%青霉素 - 链霉素的DMEM) 孔板。
      2. 转染前2小时,在80%细胞汇合时,每孔更换2ml生长培养基
      3. 稀释2微克的HBV 1.2mer复制子在125微升的Opti-MEM和轻轻地。
      4. 稀释4微升Lipofectamine 2000在125微升Opti-MEM,轻轻地轻弹。 然后,在室温下孵育5分钟
      5. 孵育5分钟后,合并稀释的HBV 1.2mer复制子 与稀释的Lipofectamine 2000并通过吸移轻轻混合。 然后, 在室温下孵育20分钟
      6. 4〜6小时后 转染,每孔更换2ml生长培养基 适当浓度的抗HBV药物(见图2)
      7. 更换 生长培养基用抗HBV药物每天4天,然后 通过在3,000rpm离心1小时收获在1.5ml管中的细胞 min。

    2. 核心颗粒相对HBV DNA的提取
      1. 加入100微升冷HEPES裂解缓冲液的细胞沉淀,并在冰上孵育20分钟
      2. 在4℃下以13,000rpm离心裂解物10分钟并转移 上清液(细胞裂解物,细胞质级分)装入新的1.5ml管中
      3. 通过移液混合5.67μl核酸酶缓冲液(I),并在37°C水浴中孵育30分钟。
      4. 简要旋转,加入40μl的26%PEG溶液,孵育2小时   在冰上(每30分钟涡旋)并在13,000rpm离心30分钟   在4℃
      5. 弃去上清液并在4℃下以13,000rpm离心5分钟
      6. 完全除去上清液(使用移液管和KIMTECH科学刮水器)
      7. 加入100μl核酸酶缓冲液(II),并物理重悬核心颗粒
      8. 在37℃水浴中孵育20分钟,并短暂旋转
      9. 加入282.5μl的0.5%SDS溶液和12.5μl蛋白酶K(20 mg/ml)并在37℃水浴中孵育2〜4小时(每30℃反转) min)。
      10. 离心后,加入400μl苯酚 - 氯仿 - 异戊醇 酒精并剧烈涡旋10秒。 然后,以13,000rpm离心   在室温下搅拌5分钟
      11. 将上层水层转移到新的1.5 ml试管中
      12. 加入40μl的3M NaOAc和800μl的100%乙醇。 反转后 数次,在-20℃下将混合物沉淀过夜
      13. 在4℃下以13,000rpm离心30分钟,弃去上清液
      14. 加入800μl的70%乙醇,轻轻翻转
      15. 在4℃下以13,000rpm离心10分钟,弃去上清液
      16. 加入800μl的100%乙醇,轻轻翻转
      17. 在4℃下以13,000rpm离心10分钟,弃去上清液
      18. 干燥HBV DNA沉淀5〜10分钟(风干),溶于15μlTE缓冲液。 将HBV DNA储存在4°C直至使用。

    3. Southern印迹测定(凝胶转移)
      1. 在89%的0.8%琼脂糖凝胶上分离HBV DNA(步骤B,步骤18)2小时
      2. 电泳后,将凝胶转移至托盘,加入转移溶液(I)以浸泡凝胶
      3. 在振荡器上孵育10分钟,并用蒸馏水冲洗凝胶
      4. 加入转移溶液(II),并在摇床上孵育45分钟
      5. 用蒸馏水冲洗并加入转移溶液(III)
      6. 在振荡器上孵育30分钟,并用蒸馏水冲洗凝胶
      7. 使用毛细管将凝胶转移至Hybond-N +膜过夜   方法(图1),并烘烤膜固定HBV DNA 2小时 80℃干烤箱。
      8. 将膜置于杂交瓶中 用25ml杂交溶液并孵育膜4小时 42℃杂交室(预杂交步骤)。

    4. HBV探针的构建
      1. 为了构建HBV特异性探针的模板,消化HBV 1.2mer 在37℃下用< em> Aat II和< em> Hind III< (Ahn et al。,2015)。
      2. 为了合成HBV特异性探针,将48ng模板(步骤8)和80ng随机引物混合在50μl蒸馏水中。
        注意:步骤10应在预杂交步骤中进行。
      3. 在100℃下加热探针混合物5分钟,在冰上快速冷却5分钟
      4. 离心后,加入5ul 10x Klenow缓冲液,2.5μldNTPs 混合物(2.5mM,除了dCTP),5μl的32P标记的dCTP(50μCi= 5 μl/标记)和1μl的探针混合物中的Klenow片段
      5. 在37℃下孵育探针混合物30分钟
      6. 使用快速旋转柱进行放射性标记的DNA纯化,去除未标记的探针。

    5. Southern印迹测定(膜杂交)
      1. 32 P标记的HBV替代预热的25ml杂交溶液 探针并在42℃杂交室中孵育过夜
      2. 杂交步骤后,用洗涤溶液(I)在63℃杂交室中洗涤膜15〜20分钟。
        注意:如果需要更多的洗涤步骤,请重复用洗涤液洗涤 (II)。 步骤9〜16应在无线电同位素室中进行。
      3. 将膜暴露于膜并在-80℃深冷冻箱中孵育过夜。 然后,显影膜。

    代表数据



    图1.用于Southern印迹的毛细管转移方法方案


    图2.耐药性HBV的体外易感性测定(Kim等人,2014)。耐药性HBV的复制是 通过Southern印迹进行分析。 TDF和ETV用如下表(下面)的浓度处理。

    食谱

    1. HEPES裂解缓冲液
      10mM Hepes(pH7.5) 100 mM NaCl
      1mM EDTA
      1%NP-40
    2. 核酸酶缓冲液(I)
      10mM CaCl 2
      12mM MgCl 2/
      DNaseI 0.5单位
      绿豆核酸酶7.5单位
    3. 26%PEG溶液
      1.2 M NaCl
      60 mM EDTA
      30%蔗糖 26%PEG 8000
    4. 核酸酶缓冲液(II)
      10mM Tris(pH7.5) 8mM CaCl 2
      6mM MgCl 2/v/v DNaseI 2单位
      绿豆核酸酶3单位
    5. 0.5%SDS溶液
      25mM Tris(pH7.5) 10 mM EDTA
      100 mM NaCl
      0.5%SDS
    6. 0.1M磷酸钠缓冲液(pH7.0) 57.7ml的1M Na 2 HPO 4
      42.3ml的1M NaH 2 PO 4·dub/d
    7. 转移溶液(I)
      0.25 N HCl
      0.6 M NaCl
    8. 转移溶液(II)
      0.5 M NaOH
      1 M NaCl
    9. 转移溶液(III)
      1 M Tris(pH 7.5〜8.0)
      1 M NaCl
    10. 杂交溶液
      甲酰胺25ml
      20x SSC 12.5ml
      0.1M磷酸钠缓冲液(pH7.0)2.5ml
      0.5M EDTA 0.2ml
      50x Denhardt溶液3ml
      10%SDS 1ml
      鲑精DNA(10mg/ml)1ml
      蒸馏水4.8ml
    11. 洗涤溶液(I)
      2x SSC
      0.1%SDS
    12. 洗涤溶液(II)
      0.5x SSC
      0.1%SDS

    致谢

    这项研究由Konkuk大学支持。

    参考文献

    1. A,SH,Park,YK,Park,ES,Kim,JH,Kim,DH,Lim,KH,Jang,MS,Choe,WH,Ko,SY,Sung,IK,Kwon,SY和Kim,KH 。 乙型肝炎病毒聚合酶rtA181T突变对复制和耐药性的影响可能受表面基因重叠变化的影响。 J Virol 88(12):6805-6818。
    2. Ahn,S.H.,Park,Y.K.and Kim,K。(2015)。 将患者分离的HBV RT序列引入和测序到HBV 1.2-mer复制子中。生物协议 5(8):e1449。
    3. Kim,J.H.,Park,Y.K.,Park,E.S.and Kim,K.H。(2014)。 耐药性乙型肝炎病毒的分子诊断和治疗世界J Gastroenterol 20(19):5708-5720。
    4. 本发明的另一个方面涉及一种用于治疗癌症的方法,所述方法包括以下步骤:(1) 。 鉴定和表征从慢性乙型肝炎患者分离的乙型肝炎病毒clevudine抗性突变体。 a> J Virol 84(9):4494-4503。
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引用: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.
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