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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   

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Jia Li
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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), Hepatocyte(肝细胞), Infection(感染), Zhr(zhr)

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

  1. Hepatocyte culture and infection with HBV
    1. HBV preparation and measurement of HBV virus titers:
      1. Seed Huh7 cells to 10-cm dish as 1 x 106 cells/dish with DMEM medium supplemented with 10% FBS and cultured at 37 °C in a 5% CO2-incubator.
      2. After 24 h culture, per dish of Huh7 cells are transfected with 10 μg HBV plasmid (pUC19-HBV-C) in Opti-MEM I reduced-serum medium by FuGENE 6 transfection reagent following the manufacturer’s protocol.
      3. At 3 days after transfection, harvest the media in 50 ml tube and clear by centrifugation for 5 min at 5,000 rpm at 4 °C. The supernatant contains the HBV can be stocked at -80 °C until use for infection.
      4. For detection the HBV DNA copies of the harvested supernatant, 100 μl supernatant mixed with 1 μl MgOAc (600 mM), 0.5 μl RNase A (20 mg/ml), 1 μl DNase I (1 unit/μl) and incubate for 3 h at 37 °C to remove the HBV plasmid. The reaction is terminated by adding 2 μl EDTA (0.5 M, pH 8.0) and incubation for 10 min at 65 °C. Then the HBV DNA is extracted using QIAamp DNA Blood Mini Kit, and HBV titers are determined by qPCR as described below (Procedure C. qPCR for quantification of HBV DNA).
    2. Seed cells for HBV infection:
      PHHs are purchased from PhoenixBio Co., Ltd. (Hiroshima, Japan) and seeded on biocoat collagen I cellware 12-well plate with 1 ml of PHHs culture medium as 1 x 105 cells/well. HepG2-hNTCP-C4 cells are generated by Drs. Takaji Wakita and Koichi Watashi (Iwamoto et al., 2014), and seeded on 12-well tissue culture plate with 1 ml of HepG2-hNTCP-C4 culture medium as 1x105 cells/well. The cells are cultured at 37 °C in a 5% CO2-incubator.
    3. HBV infection:
      After 24 h, PHHs or HepG2-hNTCP-C4 cells are incubated for 24 h at 37 °C with HBV stock aliquots containing the appropriate number of genome equivalents (GEq), which are diluted with 1 ml of culture medium supplemented with 4% PEG 8000. At the end of the incubation, cells are washed three times with culture medium, and harvested for analysis.
      Note: PHHs or HepG2-hNTCP-C4 cells are infected at 10 or 100 GEq/cell, respectively.

  2. Encapsidated HBV DNA extraction
    1. Remove culture medium, wash cells twice with cold PBS.
    2. Add lysis buffer 300 μl/well.
    3. Shake with 200 rpm/min for 20 min at cold room (4 °C) to disrupt cells.
    4. Transfer all cell lysates to new 1.5 ml tubes.
    5. Pellet nuclei and insoluble fractions by centrifugation (15,000 rpm) at 4 °C for 2 min.
    6. Harvest the supernatants to new 1.5 ml tubes (on ice).
    7. Make a mixture in the following order:

      Mix order

      Volume (μl)
      1
      Nuclease free-H2O
      20
      2
      Sample supernatant
      250
      3
      100 mM MgOAc
      18
      4
      RNase A (20 mg/ml)
      2
      5
      DNase I (1 unit/μl)
      10

      Total
      300

    8. Incubate the mixture sample at 37 °C for 6 h. (After this step, the sample can be kept at 4 °C).
    9. Add 30 μl EDTA (100 mM, pH 8.0) and incubate for 15 min at 65 °C to stop the reaction.
    10. Centrifuge for 10 min at 15,000 rpm at 4 °C, and then harvest all supernatants to new tubes.
    11. Make a mixture in the following order:

      Mix order

      Volume (μl)
      1
      Sample supernatant
      330
      2
      10% SDS
      40
      3
      Nuclease free-H2O
      18
      4
      5M NaCl
      8
      5
      Proteinase K (20 mg/ml)
      4

      Total
      400

    12. Incubate the mixture sample for 2 h at 55 °C to digest the HBV core protein and release the viral genome from core particles.
    13. Add 400 μl phenol:chloroform:isoamyl alcohol (25:24:1) and vortex thoroughly for approximately 1 min.
    14. Centrifuge for 5 min at 15,000 rpm at room temperature.
    15. Carefully transfer the upper layer to a new tube.
    16. Add 400 μl chloroform and vortex thoroughly for approximately 1 min.
    17. Centrifuge for 5 min at 15,000 rpm at room temperature.
    18. Carefully transfer the upper layer to a new tube.
    19. Add 40 μl NaOAc (3 M, pH 5.2), 1 μl glycogen (20 μg/μl) and 400 μl isopropanol.
    20. Simply vortex and place on ice for 20 min.
    21. Centrifuge for 20 min at 15,000 rpm at 4 °C.
    22. Carefully remove the supernatant without disturbing the pellet.
    23. Add 500 μl of 70% ethanol, centrifuge the sample for 10 min at 15,000 rpm at 4 °C.
    24. Carefully remove the supernatant as much as possible.
    25. Dry the pellet for 20 min at room temperature.
    26. Resuspend the pellet with 100 μl Nuclease free-H2O and briefly spin to collect the sample. (The purified encapsidated HBV DNA can be stock at -20 °C.)

  3. qPCR for quantification of HBV DNA
    1. Prepare HBV DNA standard sample:
      The HBV plasmid (pUC19-HBV) is subjected to a 10-fold serial dilutions in Nuclease free-H2O ranging from 0.2 x 103 to 109 copies/μl (1 μg HBV plasmid (pUC19-HBV) contains 1.46 x 1011 DNA copies, http://www.endmemo.com/bio/dnacopynum.php). And these diluted samples are used to construct a standard curve for the quantification of HBV DNA.
    2. Make a 100-fold dilution of the purified encapsidated HBV DNA samples with Nuclease free-H2O for each qPCR template.
    3. Set up the following reaction mixtures for qPCR analysis to measure the copy number of HBV DNA (for one sample):

      Nuclease free-H2O
      3.8 μl
      2x SYBR Premix Ex Taq
      10 μl
      50x ROX reference dye
      0.4 μl
      Forward primer (10 μM)
      0.4 μl
      Reverse primer (10 μM)
      0.4 μl
      Diluted HBV DNA sample or standard DNA sample
      5 μl
      Total
      20 μl

      Note: Use 5 μl of nuclease free-H2O instead of DNA sample as negative control. Each sample is prepared in triplicate.
    4. The PCR plate is covered with an adhesive transparent cover, and then centrifuged shortly.
    5. Set the plate in the real-time instrument and start the real-time PCR following the program as below:

      Holding Stage
      95 °C
      10 sec

      1 cycle
      Cycling Stage
      95 °C
      5 sec

      45 cycles
      60 °C
      30 sec
      Data collection
      Melt Curve Stage
      95 °C
      15 sec

      1 cycle
      60 °C
      60 sec
      Data collection during 60 °C → 95 °C
      95 °C
      15 sec

Recipes

  1. PHHs culture media
    DMEM supplemented with 10% FBS
    100 U/ml penicillin
    100 μg/ml streptomycin
    20 mM HEPES
    44 mM NaHCO3
    15 μg/ml L-proline
    0.25 μg/ml insulin
    50 nM dexamethazone
    5 ng/ml epidermal growth factor (EGF)
    0.1 mM Asc-2P
    2% dimethyl sulfoxide (DMSO)
  2. HepG2-hNTCP-C4 culture media
    DMEM/F-12+GlutaMax supplemented with 10 mM HEPES
    200 U/ml penicillin
    200 μg/ml streptomycin
    10% FBS
    50 μM hydrocortisone
    5 μg/ml insulin in the presence of 400 μg/ml G418
  3. Lysis buffer
    1% NP-40
    1 mM EDTA
    50 mM Tris-HCl (pH 7.4)

Acknowledgments

This protocol, which was adapted from Sato et al. (2015), is based on the earlier works by Turelli et al. (2004) and Sugiyama et al. (2006). This was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research [A] [25253030] to A.T., Grant-in-Aid for Scientific Research on Innovative Areas [25115502, 23112701] to A.T., Grant-in-Aid for Young Scientists [B] [25870015] to S.S.)

References

  1. Iwamoto, M., Watashi, K., Tsukuda, S., Aly, H. H., Fukasawa, M., Fujimoto, A., Suzuki, R., Aizaki, H., Ito, T., Koiwai, O., Kusuhara, H. and Wakita, T. (2014). Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Biophys Res Commun 443(3): 808-813.
  2. Sugiyama, M., Tanaka, Y., Kato, T., Orito, E., Ito, K., Acharya, S. K., Gish, R. G., Kramvis, A., Shimada, T., Izumi, N., Kaito, M., Miyakawa, Y. and Mizokami, M. (2006). Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens. Hepatology 44(4): 915-924.
  3. Sato, S., Li, K., Kameyama, T., Hayashi, T., Ishida, Y., Murakami, S., Watanabe, T., Iijima, S., Sakurai, Y., Watashi, K., Tsutsumi, S., Sato, Y., Akita, H., Wakita, T., Rice, C. M., Harashima, H., Kohara, M., Tanaka, Y. and Takaoka, A. (2015). The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus. Immunity 42(1): 123-132.
  4. Turelli, P., Mangeat, B., Jost, S., Vianin, S. and Trono, D. (2004). Inhibition of hepatitis B virus replication by APOBEC3G. Science 303(5665): 1829.
  5. Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., Huang, Y., Qi, Y., Peng, B., Wang, H., Fu, L., Song, M., Chen, P., Gao, W., Ren, B., Sun, Y., Cai, T., Feng, X., Sui, J. and Li, W. (2012). Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1: e00049.

简介

人肝癌细胞系如HepG2,Huh7和HLE由于缺乏HBV受体而不能感染乙型肝炎病毒(HBV)。 HBV基因组的转染迄今为止被称为模拟HBV感染的工具。 然而,由于牛磺胆酸钠共转运多肽(NTCP)被鉴定为HBV的功能性受体(Yan等人,2012),已经使用用用于NTCP表达的质粒稳定转染的肝细胞细胞系 为HBV感染。 该方案设计用于在表达人类肝细胞细胞系HepG2的表达NTCP(HepG2-hNTCP-C4细胞; Iwamoto等人,2014)或原代人肝细胞(PHH)的HBV感染。 在本节中,我们还描述了用于评估HBV感染的方法之一:细胞内衣壳化的HBV DNA的定量。

关键字:乙型肝炎病毒, 肝细胞, 感染, zhr

材料和试剂

材料

  1. 0.1-10μl移液管吸头(Thermo Fisher Scientific,目录号:QSP#TF104)
  2. 1-200μl和100-1,000μl移液管吸头(Corning,目录号:分别为4845和4846)
  3. Falcon 12孔组织培养板(Corning,目录号:353043)
  4. Biocoat胶原I细胞制品12孔板(Corning,目录号:356500)
  5. 1.5ml和2.0ml微量离心管(Corning,目录号:MCT-150-A和MCT-200-C)
  6. 15ml和50ml离心管(分别为Corning Incorporated,目录号:352096和352070)
  7. 96孔快板(NIPPON Genetics,目录号:38801)

试剂

  1. 肝细胞培养和HBV感染
    1. 原代人肝细胞(PHH)(PhoenixBio Co.)
    2.   HepG2-hNTCP-C4细胞(Takaji Wakita和Koichi Watashi博士,Department of Virology II,National Institute of Infectious Diseases,Tokyo,Japan; ?Iwamoto等人,2014)
    3. Dulbecco's Modified Eagle's Medium(DMEM)(NISSUI PHARMACEUTICAL,目录号:05919)
    4. DMEM/F-12 + GlutaMax(Life Technologies,目录号:31331-028) 注意:目前,它是"Thermo Fisher Scientific,Gibco?,目录号: 31331-028 "。
    5. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(Life Technologies,目录号:15630-080)
      注意:目前,它是"Thermo Fisher Scientific,Gibco?,目录号: 15630-080 "。
    6. 胎牛血清(FBS)(Life Technologies,目录号:10437-028)
      注意:目前,它是"Thermo Fisher Scientific,Gibco?,目录号: 10437-028 "。
    7. G418(Nacalai tesque,目录号:09380-86)
    8. 磷酸盐缓冲盐水(PBS)(pH 7.4)
    9. HBV质粒(pUC19-HBV,基因型C)(Dr. Yasuhito Tanaka,Department ?名古屋市立大学研究生院病毒学与肝脏科 医学科学,名古屋,日本; Sugiyama ,2006)
    10. FuGENE 6转染试剂(Promega公司,目录号:E2692)
    11. Opti-MEM I还原血清培养基(Life Technologies,目录号:31985-070)
      注意:目前,它是"Thermo Fisher Scientific,Gibco?,目录号: 31985-070 "。
    12. QIAamp DNA Blood Mini Kit(QIAGEN,目录号:51104)
    13. 聚乙二醇8000(PEG8000)(Sigma-Aldrich,目录号:81268)
    14. PHHs培养基(参见食谱)
    15. HepG2-hNTCP-C4培养基(参见配方)

  2. 包膜HBV DNA提取
    1. 核酸酶游离-H 2 O 2 /
    2. 三(羟甲基)氨基甲烷(Tris)(Nacalai tesque,目录号:35406-91)
    3. NP-40(Nacalai tesque,目录号:25223-75)
    4. 乙酸镁(MgOAc)(Wako Pure Chemical Industries,Siyaku,目录号:130-00095)
    5. 乙二胺四乙酸(EDTA)(Wako Pure Chemical Industries,Siyaku,目录号:345-01865)
    6. 蛋白酶K(Life Technologies,目录号:25530-015)
      注意:目前,它是"Thermo Fisher Scientific,Invitrogen?,目录号:
    7. 十二烷基硫酸钠(SDS)(Wako Pure Chemical Industries,Siyaku,目录号:191-07145)
    8. 氯化钠(NaCl)(Nacalai tesque,目录号:31320-05)
    9. 脱氧核糖核酸酶I(DNase I)(Promega Corporation,目录号:M6101A)
    10. 核糖核酸酶A(RNase A)(Life Technologies,目录号:12091-021)
      注意:目前,它是"Thermo Fisher Scientific,Invitrogen?,目录号: 12091-021 "。
    11. 苯酚:氯仿:异戊醇(25:24:1)(Sigma-Aldrich,目录号:P2069)
    12. 氯仿(KANTO CHEMICAL,目录号:07278-00)
    13. 乙酸钠(NaOAc)(Wako Pure Chemical Industries,Siyaku,目录号:198-01055)
    14. 糖原(Roche Diagnostics,目录号:10901393001)
    15. 异丙醇(Nacalai tesque,目录号:29113-53)
    16. 乙醇(99.5%)(Nacalai tesque,目录号:14713-95)
    17. 裂解缓冲液(见配方)

  3. 用于定量HBV DNA的qPCR
    1. SYBR Premix Ex Taq(2x)(Tli RNase H Plus)(Takara Bio Company,目录号:RR420)
    2. ROX参比染料(50x)(Takara Bio公司,目录号:A9701A)
    3. 通过定量PCR扩增衣壳化HBV DNA的引物 正向:5'-CTTCATCCTGCTGCTATGCCT-3'
      反向:5'-AAAGCCCAGGATGATGGGAT-3'
      (产物长度:222bp,产物Tm:83.5℃)

设备

  1. 37℃和5%CO 2细胞培养箱(WAKENBTECH CO。,目录号:9000EX)中。
  2. 移液管(PIPETMAN P2,P20和P1000)(Gilson Scientific,目录号:F144801,F123600和F123602)
  3. 高速冷冻微型离心机(KUBOTA Corporation,目录号:3500)
  4. 涡旋混合器(Labnet Internationa,目录号:vx100)
  5. Shaker(Tokyo Rikakikai Co.,EYELA,目录号:MMS-110)
  6. 双铝块槽(SCINICS CORPORATION,目录号:ALB-301)
  7. ABI StepOnePlus TM 实时PCR系统(Life Technologies,目录号:4379216)
    注意:目前,它是"Thermo Fisher Scientific,Applied Biosystems?,目录号: "4379216
  8. qPCR粘合密封(NIPPON Genetics,目录号:4Ti-0560)

程序

  1. 肝细胞培养和HBV感染
    1. HBV制备和HBV病毒滴度的测量:
      1. 种子Huh7 ?细胞至10-cm培养皿,1×10 6个细胞/培养皿,补充DMEM培养基 ?用10%FBS稀释,并在37℃,5%CO 2培养箱中培养。
      2. 后 24 h培养,每皿用10μgHBV转染Huh7细胞 质粒(pUC19-HBV-C)在Opti-MEM I还原血清培养基中通过FuGENE 6 根据制造商的方案转染。
      3. 在3 ?转染后天,在50ml管中收获培养基并通过 在4℃下以5,000rpm离心5分钟。上清液含有 可以将HBV储存在-80℃直到用于感染。
      4. 对于 检测收获的上清液的HBV DNA拷贝,100μl 上清液与1μlMgOAc(600mM),0.5μlRNA酶A(20mg/ml)混合, ?μlDNA酶I(1单位/μl),37°C孵育3小时以去除HBV 质粒。通过加入2μlEDTA(0.5M,pH 8.0)终止反应, 并在65℃温育10分钟。然后使用提取HBV DNA QIAamp DNA Blood Mini Kit,以及HBV滴度由qPCR确定 (程序C.用于定量HBV DNA的qPCR)。
    2. HBV感染的种子细胞:
      PHH购自PhoenixBio Co.,Ltd。(Hiroshima,Japan)和 接种在具有1ml PHH的biocoat胶原I细胞器12孔板上 培养基作为1×10 5个细胞/孔。产生HepG2-hNTCP-C4细胞 ?由Drs。 Takaji Wakita和Koichi Watashi(Iwamoto等人,2014),以及 接种在具有1ml HepG2-hNTCP-C4的12孔组织培养板上 培养基以1×10 5个细胞/孔。细胞在37℃下在37℃下培养 ?5%CO 2 - 培养基
    3. HBV感染:
      24小时后,PHHs或 将HepG2-hNTCP-C4细胞在37℃下与HBV原液温育24小时 含有适当数目的基因组当量(GEq)的等分试样, 其用1ml补充有4%PEG的培养基稀释 在温育结束时,细胞用洗涤三次 培养基,并收获用于分析。
      注意:PHH或HepG2-hNTCP-C4细胞分别以10或100 GEq /细胞感染。

  2. 包膜HBV DNA提取
    1. 取出培养基,用冷PBS洗涤细胞两次
    2. 加入裂解缓冲液300μl/孔
    3. 在冷室(4℃)下以200rpm/min摇动20分钟以破坏细胞
    4. 转移所有细胞裂解物到新的1.5毫升管。
    5. 通过在4℃下离心(15,000rpm)2分钟沉淀颗粒核和不溶性级分。
    6. 收获上清液到新的1.5毫升管(在冰上)
    7. 按以下顺序混合:

      混合顺序

      体积(μl)
      1
      核酸酶游离-H 2 O 2 / 20
      2
      样品上清
      250
      3
      100mM MgOAc
      18
      4
      核糖核酸酶A(20mg/ml) 2
      5
      DNase I(1单位/μl)
      10

      总计
      300

    8. 孵育混合物样品在37℃下6小时。 (此步骤后,样品可保持在4℃)
    9. 加入30μlEDTA(100mM,pH8.0)并在65℃下孵育15分钟以终止反应
    10. 在4℃下以15,000rpm离心10分钟,然后将所有上清液收获到新管中
    11. 按以下顺序混合:

      混合顺序

      体积(μl)
      1
      样品上清液
      300
      2
      10%SDS
      40
      3
      核酸酶游离-H 2 O 2 / 18
      4
      5M NaCl
      8
      5
      蛋白酶K(20mg/ml)
      4

      总计
      400

    12. 在55°C孵育混合物样品2小时以消化HBV核心 ?蛋白质并从核心颗粒释放病毒基因组
    13. 加入400μl苯酚:氯仿:异戊醇(25:24:1),彻底涡旋约1分钟
    14. 在室温下以15,000rpm离心5分钟。
    15. 小心地将上层转移到新管。
    16. 加入400μl氯仿,彻底涡旋约1分钟
    17. 在室温下以15,000rpm离心5分钟。
    18. 小心地将上层转移到新管。
    19. 加入40μlNaOAc(3M,pH 5.2),1μl糖原(20μg/μl)和400μl异丙醇。
    20. 只需涡旋并放在冰上20分钟。
    21. 在4℃下以15,000rpm离心20分钟。
    22. 小心地清除上清液而不干扰沉淀
    23. 加入500μl的70%乙醇,在4℃下以15,000rpm离心样品10分钟。
    24. 小心地尽可能除去上清液。
    25. 在室温下干燥沉淀20分钟。
    26. 用100μl无核酸酶的H 2 O 2重悬沉淀并短暂旋转 ?以收集样品。 (纯化的衣壳化的HBV DNA可以是储备液 在-20℃。

  3. 用于定量HBV DNA的qPCR
    1. 准备HBV DNA标准样品:
      HBV质粒(pUC19-HBV)是 在无核酸酶游离-H 2 O范围内进行10倍系列稀释 从0.2×10 3至10 10拷贝/μl(1μgHBV质粒(pUC19-HBV)含有 1.46×10 11个DNA拷贝,http://www.endmemo.com/bio/dnacopynum.php)。和 这些稀释的样品用于构建标准曲线 HBV DNA的定量。
    2. 对于每个qPCR模板,用无核酸酶的H 2 O 2对纯化的壳体化的HBV DNA样品进行100倍稀释。
    3. 设置以下反应混合物用于qPCR分析以测量HBV DNA的拷贝数(对于一个样品):

      核酸酶游离-H 2 O 2 / 3.8μl
      2x SYBR Premix Ex Taq
      10微升
      50x ROX参比染料
      0.4μl
      正向引物(10μM)
      0.4μl
      反向引物(10μM)
      0.4μl
      稀释的HBV DNA样品或标准DNA样品
      5微升
      总共
      20微升

      注意:使用5μl的核酸酶游离-H 2 O而不是DNA样品作为阴性对照。每个样品一式三份制备。
    4. PCR板用粘性透明盖覆盖,然后短时间离心
    5. 在实时仪器中设置板,并按照以下程序启动实时PCR:

      控制阶段
      95°C
      10秒

      1个周期
      循环阶段
      95°C
      5秒

      45个周期
      60°C
      30秒
      数据收集
      熔体曲线阶段
      95°C
      15秒

      1个周期
      60°C
      60秒
      60°C→95°C时的数据收集
      95°C
      15秒

食谱

  1. PHHs培养基
    补充有10%FBS的DMEM 100 U/ml青霉素
    100μg/ml链霉素 20 mM HEPES
    44mM NaHCO 3/v/v 15μg/ml L-脯氨酸 0.25μg/ml胰岛素 50nM地塞米松 5ng/ml表皮生长因子(EGF) 0.1mM Asc-2P 2%二甲基亚砜(DMSO)
  2. HepG2-hNTCP-C4培养基
    补充有10mM HEPES的DMEM/F-12 + GlutaMax 200 U/ml青霉素
    200μg/ml链霉素 10%FBS
    50μM氢化可的松
    在400μg/ml G418存在下5μg/ml胰岛素
  3. 裂解缓冲液
    1%NP-40
    1mM EDTA
    50mM Tris-HCl(pH7.4)

致谢

从Sato等人(2015)改编的该方案基于Turelli等人(2004)和Sugiyama等人(2004)的早期着作, 。(2006)。这得到了来自日本教育,文化,体育,科学和技术部的资助(授予科学研究资助[A] [25253030] to AT,Grant-in-Aid for Scientific Research on Innovative Areas [25115502,23112701] to AT,Grant-in-Aid for Young Scientists [B] [25870015]至SS)

参考文献

  1. Iwamoto,M.,Watashi,K.,Tsukuda,S.,Aly,HH,Fukasawa,M.,Fujimoto,A.,Suzuki,R.,Aizaki,H.,Ito,T.,Koiwai,O.,Kusuhara ,H。和Wakita,T。(2014)。 使用过表达膜转运蛋白NTCP的HepG2细胞评估和鉴定乙型肝炎病毒进入抑制剂。 Biochem Biophys Res Commun 443(3):808-813
  2. Sugiyama,M.,Tanaka,Y.,Kato,T.,Orito,E.,Ito,K.,Acharya,SK,Gish,RG,Kramvis,A.,Shimada,T., M.,Miyakawa,Y.and Mizokami,M。(2006)。 乙型肝炎病毒基因型对病毒DNA和抗原的细胞内和细胞外表达的影响。 a> Hepatology 44(4):915-924
  3. Sato,S.,Li,K.,Kameyama,T.,Hayashi,T.,Ishida,Y.,Murakami,S.,Watanabe,T.,Iijima,S.,Sakurai,Y.,Watashi, Tsutsumi,S.,Sato,Y.,Akita,H.,Wakita,T.,Rice,CM,Harashima,H.,Kohara,M.,Tanaka,Y.and Takaoka, RNA传感器RIG-I双重作为乙型肝炎病毒的先天传感器和直接抗病毒因子。 免疫力 42(1):123-132。
  4. Turelli,P.,Mangeat,B.,Jost,S.,Vianin,S。和Trono,D。(2004)。 APOBEC3G对乙型肝炎病毒复制的抑制 科学 303(5665):1829.
  5. 中国农业大学学报(农业与生命科学版),北京农业大学学报(农业与生命科学版),北京农业大学学报(农业与生命科学版) Fu,L.,Song,M.,Chen,P.,Gao,W.,Ren,B.,Sun,Y.,Cai,T.,Feng,X.,Sui,J.and Li, 2012)。 牛磺胆酸钠共转运多肽是人乙型和丙型肝炎病毒的功能性受体。 em> Elife 1:e00049。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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