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HBV Infection in Human Hepatocytes and Quantification of Encapsidated HBV DNA

人肝细胞的HBV感染和壳体化HBV DNA的量化

KL Kai Li
SS Seiichi Sato
Akinori Takaoka Akinori Takaoka

发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1717 浏览次数: 11286

评审: Jia LiRamalingam BethunaickanAnonymous reviewer(s)

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Abstract

Human hepatic cancer cell lines such as HepG2, Huh7, and HLE cannot get infected with Hepatitis B virus (HBV) due to lack of an HBV receptor(s). Transfection with HBV genome has so far been referred as a tool to mimic HBV infection. However, since sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HBV (Yan et al., 2012), hepatocyte cell lines that were stably transfected with a plasmid for NTCP expression have been used for HBV infection. This protocol is designed for infection with HBV in human hepatocyte cell line HepG2 expressing NTCP (HepG2-hNTCP-C4 cells; Iwamoto et al., 2014) or primary human hepatocytes (PHHs). In this section, we also describe one of the methods for the assessment of HBV infection: Quantification of the intracellular encapsidated HBV DNA.

Keywords: Hepatitis B virus (乙型肝炎病毒) search Hepatocyte (肝细胞) search Infection (感染) search Zhr (zhr) search

Materials and Reagents

Materials

  1. 0.1-10 μl pipet tips (Thermo Fisher Scientific, catalog number: QSP#TF104 )
  2. 1-200 μl and 100-1,000 μl pipet tips (Corning, catalog numbers: 4845 and 4846 , respectively)
  3. Falcon 12-well tissue culture plate (Corning, catalog number: 353043 )
  4. Biocoat collagen I cellware 12-well plate (Corning, catalog number: 356500 )
  5. 1.5 ml and 2.0 ml microcentrifuge tubes (Corning, catalog numbers: MCT-150-A and MCT-200-C , respectively)
  6. 15 ml and 50 ml centrifuge tubes (Corning Incorporated, catalog numbers: 352096 and 352070 , respectively)
  7. 96-well fast plate (NIPPON Genetics, catalog number: 38801 )

Reagents

  1. Hepatocyte culture and infection with HBV
    1. Primary human hepatocytes (PHHs) (PhoenixBio Co.)
    2. HepG2-hNTCP-C4 cells (Drs. Takaji Wakita and Koichi Watashi, Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Iwamoto et al., 2014)
    3. Dulbecco’s Modified Eagle’s Medium (DMEM) (NISSUI PHARMACEUTICAL, catalog number: 05919 )
    4. DMEM/F-12+GlutaMax (Life Technologies, catalog number: 31331-028 )
      Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 31331-028 ”.
    5. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Life Technologies, catalog number: 15630-080 )
      Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 15630-080”.
    6. Fetal bovine serum (FBS) (Life Technologies, catalog number: 10437-028 )
      Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 10437-028”.
    7. G418 (Nacalai tesque, catalog number: 09380-86 )
    8. Phosphate-buffered saline (PBS) (pH 7.4)
    9. HBV plasmid (pUC19-HBV, genotype C) (Dr. Yasuhito Tanaka, Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Sugiyama et al., 2006)
    10. FuGENE 6 transfection reagent (Promega Corporation, catalog number: E2692 )
    11. Opti-MEM I reduced-serum medium (Life Technologies, catalog number: 31985-070 )
      Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 31985-070”.
    12. QIAamp DNA Blood Mini Kit (QIAGEN, catalog number: 51104 )
    13. Polyethyleneglycol 8000 (PEG 8000) (Sigma-Aldrich, catalog number: 81268 )
    14. PHHs culture media (see Recipes)
    15. HepG2-hNTCP-C4 culture media (see Recipes)

  2. Encapsidated HBV DNA extraction
    1. Nuclease free-H2O
    2. Tris (hydroxymethyl) aminomethane (Tris) (Nacalai tesque, catalog number: 35406-91 )
    3. NP-40 (Nacalai tesque, catalog number: 25223-75 )
    4. Magnesium acetate (MgOAc) (Wako Pure Chemical Industries, Siyaku, catalog number: 130-00095 )
    5. Ethylenediaminetetraacetic acid (EDTA) (Wako Pure Chemical Industries, Siyaku, catalog number: 345-01865 )
    6. Proteases K (Life Technologies, catalog number: 25530-015 )
      Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 25530-015”.
    7. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, Siyaku, catalog number: 191-07145 )
    8. Sodium chloride (NaCl) (Nacalai tesque, catalog number: 31320-05 )
    9. Deoxyribonuclease I (DNase I) (Promega Corporation, catalog number: M6101A )
    10. Ribonuclease A (RNase A) (Life Technologies, catalog number: 12091-021 )
      Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 12091-021”.
    11. Phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich, catalog number: P2069 )
    12. Chloroform (KANTO CHEMICAL, catalog number: 07278-00 )
    13. Sodium acetate (NaOAc) (Wako Pure Chemical Industries, Siyaku, catalog number: 198-01055 )
    14. Glycogen (Roche Diagnostics, catalog number: 10901393001 )
    15. Isopropanol (Nacalai tesque, catalog number: 29113-53 )
    16. Ethanol (99.5%) (Nacalai tesque, catalog number: 14713-95 )
    17. Lysis buffer (see Recipes)

  3. qPCR for quantification of HBV DNA
    1. SYBR Premix Ex Taq (2x) (Tli RNase H Plus) (Takara Bio Company, catalog number: RR420 )
    2. ROX reference dye (50x) (Takara Bio Company, catalog number: A9701A )
    3. Primers for amplification of encapsidated HBV DNA by quantitative PCR
      Forward: 5’-CTTCATCCTGCTGCTATGCCT-3’
      Reverse: 5’-AAAGCCCAGGATGATGGGAT-3’
      (Product length: 222 bp, product Tm: 83.5 °C)

Equipment

  1. 37 °C and 5% CO2 cell culture incubator (WAKENBTECH CO., catalog number: 9000EX )
  2. Pipettes (PIPETMAN P2, P20 and P1000) (Gilson Scientific, catalog numbers: F144801 , F123600 and F123602 , respectively)
  3. High speed refrigerated micro centrifuge (KUBOTA Corporation, catalog number: 3500 )
  4. Vortex mixer (Labnet Internationa, catalog number: vx100 )
  5. Shaker (Tokyo Rikakikai Co., EYELA, catalog number: MMS-110 )
  6. Double aluminum block bath (SCINICS CORPORATION, catalog number: ALB-301 )
  7. ABI StepOnePlus™ Real-Time PCR Systems (Life Technologies, catalog number: 4379216 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems™, catalog number: 4379216”.
  8. qPCR adhesive seal (NIPPON Genetics, catalog number: 4Ti-0560 )

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Li, K., Sato, S. and Takaoka, A. (2016). HBV Infection in Human Hepatocytes and Quantification of Encapsidated HBV DNA. Bio-protocol 6(2): e1717. DOI: 10.21769/BioProtoc.1717.

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分类

微生物学 > 微生物遗传学 > DNA > DNA 定量

微生物学 > 微生物-宿主相互作用 > 体外实验模型

微生物学 > 微生物-宿主相互作用 > 病毒

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