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Characterization of HBV Isolates from Patient Serum Samples and Cloning
病人血清样本和克隆中HBV分离体的特性描述   

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Abstract

Hepatitis B virus (HBV) mutants can lead to vaccine failure, diagnostic failure of HBV detection, increase viral replication and resistance to antiviral agents. To study the biological characteristics of these mutations may contribute to our knowledge on viral pathogenesis. Therefore, it is essential to isolate and characterize HBV strains from patients. Here we describe the experimental methods to isolate and clone HBV DNA from patient serum. The method will facilitate isolation and functional analysis of new HBV variants.

Materials and Reagents

  1. Extraction of HBV DNA from patient serum samples
    1. 1.5 ml microfuge tube (Axygen, catalog number: 17615044 )
    2. Patient serum samples
    3. Tris phenol (Beijing Shuang Xiang Da Company, catalog number: 108952 )
    4. Trichloromethane (Sinopharm Chemical Reagent Co., catalog number: 61553 )
    5. Ethanol (Sinopharm Chemical Reagent Co., catalog number: 32061 )
    6. Isopropyl alcohol (Sinopharm Chemical Reagent Co., catalog number: 32064 )
    7. Yeast RNA (Life Technologies, Ambion®, catalog number: AM7118 )
      Note: Currently, it is “Thermo Fisher Scientific, AmbionTM, catalog number: AM7118”.
    8. Sodium acetate trihydrate (NaOAc) (Sigma-Aldrich, catalog number: 236500 )
    9. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
    10. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
    11. Tris-base (Sigma-Aldrich, catalog number: T1378 )
    12. Viral lysis buffer (see Recipes)

  2. Amplification of HBV DNA by Polymerase Chain Reaction (PCR)
    1. KOD-PLUS: a high-fidelity DNA polymerase (TOYOBO CO., catalog number: KOD201 )
    2. Primers used for PCR as described below (Detailed description can refer to Figure 1)

      Name
      Type
      Sequence 5’-3’
      Position of 5’-base
      P1
      forward
      CCGGCGTCGACGAGCTCTTCT
      TTTTCACCTCTGCCTAATCA
      1821
      P2
      forward
      CCGGCGTCGACGAGCTCTTCA
      AAAAGTTGCATGGTGCTGG
      1825
      P3
      reverse
      CACTGAACAAATGGCACTAGT
      AAACTGAGCC
      699
      P4
      reverse
      GGCTCAGTTTACTAGTGCCATT
      TGTTCAGTG
      669

      Note: The underlined red letters are the restriction endonuclease binding site. P1: Sal I, Sac I; P2: Sal I, Sac I; P3: Spe I; P4: Spe I. P1/P3 are used to amplify the 2.05 kb fragment (1,821-699 bp), P2/P4 are used to amplify the 1.15 kb fragment (669-1,825 bp).

  3. Gel extraction of HBV DNA
    1. AxyPrepTM DNA Gel Extraction kit (Thermo Fisher Scientific, AxygenTM, catalog number: APGX50 )

  4. Add dATP to generate 3’ overhangs
    1. Taq DNA polymerase (TOYOBO CO., catalog number: TAP201 )
    2. dATP (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0141 )

  5. Integration of HBV DNA into pGEM®-T Vector
    1. pGEM®-T Vector System I (Promega Corporation, catalog number: A3600 )
    2. 0.5 ml microfuge tube (Axygen, catalog number: 05615119 )

  6. Transformation
    1. Competent DH5α (Takara, catalog number: 9057 )
    2. Ampicillin (Sinopharm Chemical Reagent Co., catalog number: 69523 )
    3. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: PHG0010-5G )
    4. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) (Sigma-Aldrich, catalog number: B4252-50MG )
    5. Sodium chloride (NaCl) (Sinopharm Chemical Reagent CO., catalog number: 10019318 )
    6. TRYPTONE (Oxoid Limited, catalog number: LP0042 )
    7. YEAST EXTRACT (Oxoid Limited, catalog number: LP0021 )
    8. LB medium (see Recipes)

  7. Extract HBV DNA-integrated plasmid from E.coli-DH5α
    1. E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-tek Inc., catalog number: D6942-02 )

  8. Integration of full-length HBV genome into the cloning vector pUC19
    1. Sac I (New England Biolabs, catalog number: R0156S )
    2. Sal I (New England Biolabs, catalog number: R0138S )
    3. Spe I (New England Biolabs, catalog number: R0133S )
    4. T4 DNA ligase (New England Biolabs, catalog number: M0202S )

Equipment

  1. Instrument for PCR (Biocompare, Biometra, catalog number: 070851 )
  2. Centrifuge (Eppendorf, model: 5424 )
  3. Electrophoresis system (Beijing Liuyi Biotechnology, model: DYY6C )
  4. Electronic scales (Mettler-Toledo International Inc., model: PB602-N )
    Note: Currently, it is “Mettler-Toledo International Inc., model: PB602-S ”.
  5. Bacteriological incubator (bio-equip, Shanghai Jing Hong Laboratory Instrument Co., model number: GNP-9080 )
  6. BIO IMAGING SYSTEM (Syngene, model: SYDR2/1361 )
  7. Heat block

Procedure

  1. Extraction of HBV DNA from patient serum samples
    1. Add 100 μl viral lysis buffer to 100 μl serum samples, incubate at 65 °C for 4 h.
    2. Add 125 μl Tris phenol and 125 μl Trichloromethane, vigorously vortex for 10 sec. Then, centrifuge at 13,000 rpm for 5 min at room temperature. This step can separate protein from nucleic acid and nucleic acid would be in the aqueous upper layer.
    3. Transfer aqueous upper layer into a new 1.5 ml tube.
    4. Add 20 μg Yeast RNA (as a co-precipitant here), 0.1 volume NaOAc and 0.7 volume Isopropyl alcohol. After inverting several times, precipitate the mixture overnight at -20 °C.
    5. Centrifuge at 13,000 rpm for 15 min at 4 °C and throw away the supernatant.
    6. Add 1 ml of 70% ethanol and gently invert.
    7. Centrifuge at 13,000 rpm for 5 min at 4 °C and throw away the supernatant.
    8. Dry the DNA pellet for 5~10 min (air-drying) and dissolve in 20 μl ddH2O as the PCR template.

  2. Amplification of HBV DNA by Polymerase Chain Reaction (PCR)
    1. PCR system:
      Reagent
      Volume/μl
      10x KOD buffer
      5 μl
      dNTPs (2.5 mM each)
      5 μl
      P1 (20 mM)
      1 μl
      P3 (20 mM)
      1 μl
      Template
      5 μl
      Mg2+ (25 mM)
      2 μl
      KOD_PLUS enzyme (1 U/μl)
      1 μl

      Add ddH2O to a final volume of 50 μl.
    2. PCR reaction program:
      94 °C, 5 min
      94 °C, 30 sec
      55 °C, 30 sec 40 cycles
      68 °C, 2 min
      68 °C, 10 min
      For the 1.15 kb fragment amplification, P2 and P4 primers are used and the extension proceeding of PCR reaction program should be changed from 68 °C, 2 min to 68 °C, 1 min.
    3. Add 10x DNA loading buffer to each sample and mix by pipetting. Purifying the PCR product by running on a 1% agarose gel at 100 V for 30 min.
      Note: The size of gel used here is 6 cm; the voltage/run-time would vary by the gel rig used.

  3. Gel extraction of HBV DNA
    1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. In this application, the weight of gel is regarded as equivalent to the volume. For example, 100 mg of gel is equivalent to a 100 μl volume. Transfer the gel slice into a 1.5 ml microfuge tube.
      Note: Alternatively, the gel slice can be placed into the 1.5 ml microfuge tube and then crushed with a pipette tip or other suitable device. Spin the tube for 30 sec at 12,000 x g to consolidate the gel at the bottom of the tube. Use the graduations to estimate the volume of the agarose gel.
    2. Add a 3x sample volume of Buffer DE-A.
      Note: The color of Buffer DE-A is red. This color is used to add contrast in the next step, so that any pieces of unsolubilized agarose can be visualized.
    3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75 °C until the gel is completely dissolved (typically, 6-8 min). Heat at 40 °C if low-melt agarose gel is used. Intermittent vortexing (every 2-3 min) will accelerate gel solubilization.
      IMPORTANT: Gel must be completely dissolved or the DNA fragment recovery will be reduced.
      IMPORTANT: Do not heat the gel for longer than 10 min.
    4. Add 0.5x Buffer DE-A volume of Buffer DE-B, mix. If the DNA fragment is less than 400 bp, supplement further with a 1x sample volume of isopropanol.
      Example: For a 1% gel slice equivalent to 100 μl, add the following:
      • 300 μl Buffer DE-A
      • 150 μl Buffer DE-B
      If the DNA fragment is <400 bp, you would also add:
      • 100 μl of isopropanol
      Note: The color of the mixture will turn yellow after the addition of Buffer DE-B. Please make sure the contents are a uniform yellow color before proceeding.
    5. Place a Miniprep column into a 2 ml microfuge tube (provided). Transfer the solubilized agarose from step C4 into the column. Centrifuge at 12,000 x g for 1 min.
    6. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000 x g for 30 sec.
    7. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000 x g for 30 sec.
      Note: Make sure that 95-100% ethanol has been added into Buffer W2 concentrate. Make a notation on the bottle label for future reference.
    8. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000 x g for 1 min.
      Note: Two washes with Buffer W2 are used to ensure the complete removal of salt, eliminating potential problems in subsequent enzymatic reactions, such as ligation and sequencing reaction.
    9. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000 x g for 1 min.
    10. Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided). To elute the DNA, add 25-30 μl of Eluent or deionized water to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000 x g for 1 min.
      Notes:
      1. Pre-warming the Eluent at 65 °C will generally improve elution efficiency.
      2. Deionized water can also be used to elute the DNA fragments.
      3. More detailed information can refer to the PDF protocol file attached here.

  4. Add dATP to generate 3’ overhangs
    1. The reaction system to add dATP:
      Reagent
      Volume/μl
      dATP
      1 μl
      10x Taq buffer
      1 μl
      Taq
      1 μl
      PCR product
      7 μl

    2. Incubate at 72 °C for 30 min.

  5. Integration of HBV DNA into pGEM®-T Vector
    1. Briefly centrifuge the pGEM®-T Vector tube to collect contents at the bottom of the tube.
    2. Set up ligation reactions as described below. Vortex the 2x Rapid Ligation Buffer vigorously before each use. Use 0.5 ml tubes known to have low DNA-binding capacity.

      Reagents
      Volume
      2X Rapid Ligation Buffer
      5 μl
      pGEM®-T Vector (50 ng)
      1 μl
      PCR product
      2 μl
      T4 DNA Ligase (3 Weiss units/µl)
      1 μl
      Deionized water to a final volume of
      10 μl

    3. Mix the reactions by pipetting. Incubate the reactions 1 h at room temperature. Alternatively, incubate the reactions overnight at 4 °C for the maximum number of transformants.
      Note: More detailed information can refer to the PDF protocol file attached here.

  6. Transformation
    1. Add 5 μl ligation product into 50 μl competent E.coli-DH5α, incubate on ice for 30 min.
    2. Incubate at 42 °C for 90 sec followed by transfering immediately on ice for 3 min.
    3. Add 1 ml LB medium, incubate at 37 °C shaker for 1 h.
    4. Centrifuge at 8,000 rpm for 2 min at room temperature and throw away the supernatant, use 50 μl LB medium to re-suspend the bacteria precipitate.
    5. Add 2 μl IPTG (storage concentration: 20%, m/V) and 20 μl X-gal (storage concentration: 2%, m/V) and mix by pipetting, then spread the solution on Amp resistance plate (final concentration of Amp: 50 μg/ml).
    6. Put the plate at 37 °C bacteriological incubator for 12-16 h.
    7. Choose the white clones to get the HBV DNA-integrated plasmid.

  7. Extract HBV DNA-integrated plasmid from E.coli-DH5α
    1. Isolate a single white colony from the plate (step F6), and inoculate a culture of 5 ml LB medium containing the Amp antibiotic. Incubate for ~12-16 h at 37 °C with vigorous shaking (~ 300 rpm). Use a 10-20 ml culture tube or a flask with a volume of at least 4 times the volume of the culture.
    2. Centrifuge at 10,000 x g for 1 min at room temperature.
    3. Decant or aspirate and discard the culture media.
    4. Add 250 μl Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
      Note: RNase A must be added to Solution I before use. Please see the instructions (attached here) in the Preparing Reagents section on Page 6.
    5. Transfer suspension into a new 1.5 ml microcentrifuge tube.
    6. Add 250 μl Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 min incubation may be necessary.
      Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 min. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.
    7. Add 350 μl Solution III. Immediately invert several times until a flocculent white precipitate forms.
      Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.
    8. Centrifuge at maximum speed (≥13,000 x g) for 10 min. A compact white pellet will form. Promptly proceed to the next step.
    9. Insert a HiBind® DNA Mini Column into a 2 ml Collection Tube.
    10. Transfer the cleared supernatant from step G8 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column.
    11. Centrifuge at maximum speed for 1 min.
    12. Discard the filtrate and reuse the collection tube.
    13. Add 500 μl HB Buffer.
    14. Centrifuge at maximum speed for 1 min.
    15. Discard the filtrate and reuse collection tube.
    16. Add 700 μl DNA Wash Buffer.
      Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see Page 6 (attached here) for instructions.
    17. Centrifuge at maximum speed for 1 min.
    18. Discard the filtrate and reuse the collection tube.
    19. Centrifuge the empty HiBind® DNA Mini Column for 2 min at maximum speed to dry the column matrix.
      Note: It is important to dry the HiBind® DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.
    20. Transfer the HiBind® DNA Mini Column to a clean 1.5 ml microcentrifuge tube.
    21. Add 30-100 μl Elution Buffer or sterile deionized water directly to the center of the column membrane.
      Note: The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5.
    22. Let sit at room temperature for 1 min.
    23. Centrifuge at maximum speed for 1 min.
      Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
    24. Plasmid sequencing and choose the right HBV DNA-integrated plasmid for below step.
      Note: More detailed information can refer to the PDF protocol file attached here.

  8. Integration of full-length HBV genome into the cloning vector pUC19
    1. Digest the 2.05 kb-HBV DNA-integrated plasmid with Sal I and Spe I, 1.15-HBV DNA-integrated plasmid with Sac I and Spe I, pUC19 plasmid with Sal I and Sac I. The detailed reaction system as described below.

      Reagents
      2.05 kb-HBV DNA- integrated plasmid
      1.15 kb-HBV DNA- integrated plasmid
      pUC19
      10x Digest buffer
      2 μl
      2 μl
      2 μl
      Sal I
      1 μl
      -
      1 μl
      Spe I
      1 μl
      1 μl
      -
      Sac I
      -
      1 μl
      1 μl
      Plasmid
      1 μg
      1 μg
      1 μg
      Deionized water to a final volume of
      20 μl
      20 μl
      20 μl

    2. Incubate at 37 °C for 1 h.
    3. Add 10x DNA loading buffer to each sample and mix by pipetting. Gel purify the product by running on a 1% agarose gel at 100 V for 30 min.
    4. Gel extraction of the 2.05 kb fragment, 1.15 kb fragment and ~2.7 kb linear pUC19 fragment as described in step C.
    5. Use T4 DNA ligase to integrate them at 4 °C overnight. The detailed reaction system as described below.

      Reagents
      Volume
      10x Ligation Buffer
      1 μl
      ~2.7 kb linear pUC19 fragment
      1 μl
      2.05 kb fragment
      3 μl
      1.15 kb fragment
      4 μl
      T4 DNA Ligase
      1 μl
      a final volume of
      10 μl

    6. Incubate the reactions overnight at 4 °C for the maximum number of transformants.
    7. Ligation products are transformed into the competent E.coli-DH5α as described in step F1-6.
    8. Extract recombinant plasmid from E.coli-DH5α as described step G.
    9. Identify the plasmid with Sac I and Spe I. The detailed steps can refer to H1-3.
    10. The plasmid that three fragments: 2.7 kb, 2.05 kb and 1.15 kb formed by digestion with Sac I and Spe I (Figure 2C) is the right full-length HBV genome-integrated plasmid, pUC/HBV plasmid.

Representative data


Figure 1. Schematic diagram of cloning for HBV DNA. HBV DNA was extracted from patient serum as the PCR template. Primer P1 and P3 are used to amplify the 2.05 kb-HBV DNA fragment; Primer P2 and P4 are used to amplify the 1.15 kb-HBV DNA fragment. The two fragments were ligated into pGEM®-T Vector, respectively and subjected to sequence analysis. The 2.05 kb-HBV DNA fragment and 1.15 kb-HBV DNA fragment were digested from the right recombinant plasmid and were ligated into the pUC19 plasmid to form the recombinant pUC/HBV plasmid.


Figure 2. Construction and Identification of pUC/HBV. A. Amplification of 1.15 kb HBV DNA fragment. Lane 1: Marker; Lane 2,3: samples. B. Amplification of 2.05 kb HBV DNA fragment. Lane 1: Marker; Lane 2,3: samples. C. Identification of pUC/HBV by Sac I and Spe I digestion. Lane 1: Marker; Lane 2: negative clone; Lane 3: positive clone.

Recipes

  1. Viral lysis buffer
    20 mM Tris-HCl (pH 8.0)
    10 mM EDTA
    0.1% SDS
    0.8 mg/ml proteinase K
  2. LB medium
    10 g/L NaCl
    10 g/L tryptone
    5 g/L yeast extract

Acknowledgments

This protocol was modified from the previous work by Hui Shi and Liang Cao. This study was supported by the National Nature Science Foundation of China (31200699).

References

  1. Alcantara, F. F., Tang, H. and McLachlan, A. (2002). Functional characterization of the interferon regulatory element in the enhancer 1 region of the hepatitis B virus genome. Nucleic Acids Res 30(9): 2068-2075.
  2. Cao, L., Wu, C., Shi, H., Gong, Z., Zhang, E., Wang, H., Zhao, K., Liu, S., Li, S., Gao, X., Wang, Y., Pei, R., Lu, M. and Chen, X. (2014). Coexistence of hepatitis B virus quasispecies enhances viral replication and the ability to induce host antibody and cellular immune responses. J Virol 88(15): 8656-8666.
  3. Gunther, S., Li, B. C., Miska, S., Kruger, D. H., Meisel, H. and Will, H. (1995). A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol 69(9): 5437-5444.
  4. He, J., He, L. and Yao, X. (1998). A novel method to study the full length genome of hepatitis B virus. Chin J Infect Dis 16(2): p70-72.
  5. Huang, L. R., Wu, H. L., Chen, P. J. and Chen, D. S. (2006). An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci U S A 103(47): 17862-17867.
  6. Li, L., Shen, H., Li, A., Zhang, Z., Wang, B., Wang, J., Zheng, X., Wu, J., Yang, D., Lu, M. and Song, J. (2013). Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B. Virol J 10: 214.

简介

乙型肝炎病毒(HBV)突变体可导致疫苗失败,HBV检测的诊断失败,增加病毒复制和对抗病毒剂的抗性。 研究这些突变的生物学特性可能有助于我们对病毒发病机制的了解。 因此,必须从患者中分离和表征HBV病毒株。 在这里我们描述从患者血清中分离和克隆HBV DNA的实验方法。 该方法将促进新的HBV变体的分离和功能分析。

材料和试剂

  1. 从患者血清样品中提取HBV DNA
    1. 1.5ml微量离心管(Axygen,目录号:17615044)
    2. 患者血清样品
    3. 三苯酚(北京双祥达公司,目录号:108952)
    4. 三氯甲烷(Sinopharm Chemical Reagent Co.,目录号:61553)
    5. 乙醇(Sinopharm Chemical Reagent Co.,目录号:32061)
    6. 异丙醇(Sinopharm Chemical Reagent Co.,目录号:32064)
    7. 酵母RNA(Life Technologies,Ambion ,目录号:AM7118)
      注意:目前,"Thermo Fisher Scientific,Ambion TM ,目录号:AM7118" />
    8. 醋酸钠三水合物(NaOAc)(Sigma-Aldrich,目录号:236500)
    9. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS)
    10. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
    11. Tris-base(Sigma-Aldrich,目录号:T1378)
    12. 病毒裂解缓冲液(见配方)

  2. 通过聚合酶链反应(PCR)扩增HBV DNA
    1. KOD-PLUS:高保真DNA聚合酶(TOYOBO CO。,目录号:KOD201)
    2. 用于如下所述的PCR的引物(详细描述可参考图1)

      名称
      输入
      序列5'-3'
      5'-碱基的位置
      P1
      前进
      CCGGC G TCGACGAGCTC
      TTTTCACCTCTGCCTAATCA
      1821
      P2
      前进
      CCGGC GTCGACGAGCT C
      AAAAGTTGCATGGTGCTGG
      1825
      P3
      反向
      CACTGAACAAATGGC ACTAGT
      AAACTGAGCC
      699
      P4
      反向
      GGCTCAGTTT ACTAGT GCCATT
      TGTTCAGTG
      669

      注意:带下划线的红色字母是限制性内切酶 结合位点。 P1:Sal I,Sac I; P2:Sal I,Sac I; P3:Spe I; P4:Spe I. P1/P3用于扩增2.05kb片段(1,821-699bp),P2/P4 ?用于扩增1.15kb片段(669-1,825bp)。

  3. HBV DNA的凝胶提取
    1. (Thermo Fisher Scientific,Axygen TM ,目录号:APGX50)

  4. 添加dATP以产生3'突出端
    1. Taq DNA聚合酶(TOYOBO CO。,目录号:TAP201)
    2. dATP(Thermo Fisher Scientific,Thermo Scientific ,目录号:R0141)

  5. 将HBV DNA整合到pGEM ? -T载体
    1. pGEM -T载体系统I(Promega公司,目录号:A3600)
    2. 0.5ml微量离心管(Axygen,目录号:05615119)

  6. 转换
    1. 感受态DH5α(Takara,目录号:9057)
    2. 氨苄西林(Sinopharm Chemical Reagent Co.,目录号:69523)
    3. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:PHG0010-5G)
    4. 5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷(X-gal)(Sigma-Aldrich,目录号:B4252-50MG)
    5. 氯化钠(NaCl)(Sinopharm Chemical Reagent CO。,目录号:10019318)
    6. TRYPTONE(Oxoid Limited,目录号:LP0042)
    7. YEAST EXTRACT(Oxoid Limited,目录号:LP0021)
    8. LB培养基(参见配方)

  7. 从大肠杆菌-DH5α提取HBV DNA整合的质粒
    1. 质粒DNA小试剂盒I(Omega Bio-tek Inc.,目录号:D6942-02)

  8. 全长HBV基因组整合到克隆载体pUC19
    1. I(New England Biolabs,目录号:R0156S)
    2. I(New England Biolabs,目录号:R0138S)
    3. I(New England Biolabs,目录号:R0133S)
    4. T4 DNA连接酶(New England Biolabs,目录号:M0202S)

设备

  1. PCR仪(Biocompare,Biometra,目录号:070851)
  2. 离心机(Eppendorf,型号:5424)
  3. 电泳系统(北京柳一生物技术有限公司,型号:DYY6C)
  4. 电子秤(Mettler-Toledo International Inc.,型号:PB602-N)
    注意:目前,它是"Mettler-Toledo International Inc.,型号:PB602-S"。
  5. 细菌培养箱(生物装置,上海京红实验室仪器有限公司,型号:GNP-9080)
  6. BIO IMAGING SYSTEM(Syngene,型号:SYDR2/1361)
  7. 热块

程序

  1. 从患者血清样品中提取HBV DNA
    1. 向100μl血清样品中加入100μl病毒裂解缓冲液,在65℃孵育4小时
    2. 剧烈加入125μlTris苯酚和125μl三氯甲烷 涡旋10秒。然后,在室温下以13,000rpm离心5分钟 温度。该步骤可以将蛋白质与核酸分离 核酸会在水层中。
    3. 将上层水溶液转移到新的1.5 ml管中。
    4. 加入20μg酵母RNA(此处作为共沉淀剂),0.1体积NaOAc和 ?0.7体积异丙醇。反转几次后, 将混合物在-20℃下沉淀过夜
    5. 在4℃下以13,000rpm离心15分钟,弃去上清液
    6. 加入1ml 70%乙醇,轻轻倒转
    7. 在4℃下以13,000rpm离心5分钟,弃去上清液
    8. 干燥DNA沉淀5?10分钟(空气干燥),并溶解在20μlddH 2 O中作为PCR模板。

  2. 通过聚合酶链反应(PCR)扩增HBV DNA
    1. PCR系统:
      试剂
      体积/μl
      10x KOD缓冲区
      5微升
      dNTP(各2.5mM) 5微升
      P1(20mM)
      1微升
      P3(20mM)
      1微升
      模板
      5微升
      Mg 2+(25mM)
      2微升
      KOD_PLUS酶(1U /μl)
      1微升

      加入ddH 2 O至最终体积为50μl。
    2. PCR反应程序:
      94℃,5分钟
      94℃,30秒
      55℃,30秒40个循环
      68℃,2分钟
      68℃,10分钟
      对于1.15kb片段扩增,使用P2和P4引物 PCR反应程序的延伸进程应改变 68℃,2分钟至68℃,1分钟
    3. 每个加入10x DNA加载缓冲液 样品并通过移液混合。通过在a上运行来纯化PCR产物 1%琼脂糖凝胶,100V,30分钟 注意:这里使用的凝胶尺寸为6厘米;电压/运行时间会随所使用的凝胶装置而变化。

  3. HBV DNA的凝胶提取
    1. 切除含有感兴趣的DNA片段的琼脂糖凝胶切片 与一个干净,锋利的手术刀在紫外线照明下。简要地说 将切下的凝胶切片放在吸收性毛巾上以除去残余物 缓冲。将凝胶切片转移到一块或塑料包装或称重 船。将凝胶切成小块并称重。在本应用中, 凝胶的重量被认为等于体积。例如, 100mg凝胶相当于100μl体积。转移凝胶切片 进入1.5ml微量离心管中 注意:或者,凝胶切片可以 ?置于1.5ml微量离心管中,然后用α粉碎 移液管尖端或其他合适的装置。旋管30秒,在12000 ?x g以凝固在管底部的凝胶。使用 以评估琼脂糖凝胶的体积。
    2. 添加3x样品体积的缓冲液DE-A 注意:Buffer DE-A的颜色是红色。这种颜色用于添加 对比在下一步,使任何片段的未溶解的琼脂糖 可以显示。
    3. 通过涡旋将凝胶重悬在缓冲液DE-A中。 ?在75℃加热直至凝胶完全溶解(通常为6-8℃) min)。如果使用低熔点琼脂糖凝胶,在40℃加热。间歇 涡旋(每2-3分钟)将加速凝胶溶解 重要信息:凝胶必须完全溶解或DNA片段恢复将减少。
      重要:请勿加热凝胶超过10分钟。
    4. 加入0.5x缓冲液DE-A体积的缓冲液DE-B,混合。如果DNA片段 ?小于400bp,进一步补充1×样品体积 异丙醇 示例:对于相当于100μl的1%凝胶切片,添加以下内容:
      ?300μl缓冲液DE-A
      ?150μl缓冲液DE-B
      如果DNA片段<400 bp,您还需要添加:
      ?100μl异丙醇
      注意:添加后混合物的颜色将变为黄色 缓冲液DE-B。请确保内容是均匀的黄色 然后继续。
    5. 将Miniprep柱放入2ml微量离心机中 管(提供)。将溶解的琼脂糖从步骤C4转移到 柱。以12,000 x g离心1分钟。
    6. 弃去滤液 从2ml微量离心管。将Miniprep柱返回到2 ml 微量离心管,加入500μl缓冲液W1。以12,000 xg离心机离心。 ?30秒。
    7. 弃去2ml微量离心管中的滤液。 将Miniprep柱返回到2ml微量离心管中,加入700μl 缓冲器W2。以12,000×g离心30秒。
      注意:请确保 ?95-100%乙醇已添加到缓冲液W2浓缩物中。做一个 瓶标签上的符号,供将来参考。
    8. 丢弃 滤液从2ml微量离心管中。将Miniprep栏放回原处 进入2ml微量离心管。添加第二个700μl等份的缓冲液W2 并以12,000×g离心1分钟 注意:用Buffer洗涤两次 ?W2用于确保完全去除盐,消除 在随后的酶反应,如连接中的潜在问题 和测序反应。
    9. 弃去2ml的滤液 微量离心管。将Miniprep柱放回2ml微量离心机中 管。以12,000 x g离心1分钟。
    10. 转移Miniprep 柱置入干净的1.5ml微量离心管(提供)中。为了洗脱DNA, 向膜的中心加入25-30μl的洗脱液或去离子水。 ?让它在室温下放置1分钟。以12,000 x g离心1分钟。
      注意:
      1. 在65℃下预热洗脱液通常会提高洗脱效率。
      2. 去离子水也可用于洗脱DNA片段。
      3. 更详细的信息可以参考此处的PDF协议文件。

  4. 添加dATP以产生3'突出端
    1. 添加dATP的反应体系:
      试剂
      体积/μl
      dATP
      1微升
      10x Taq缓冲区
      1微升
      Taq
      1微升
      PCR产物
      7微升

    2. 在72℃孵育30分钟。

  5. 将HBV DNA整合到pGEM ? -T载体
    1. 短暂离心pGEM -T Vector管,收集管底部的内容物。
    2. 如下所述设置连接反应。涡旋2x快速 每次使用前大力连接缓冲液。使用已知的0.5毫升管 具有低DNA结合能力
      试剂

      2X快速连接缓冲液
      5微升
      pGEM ? -T Vector(50 ng)
      1微升
      PCR产物
      2微升
      T4 DNA连接酶(3Weiss单位/μl)
      1微升
      去离子水至最终体积为
      10微升

    3. 通过吸移混合反应。在室温下孵育反应1小时 温度。或者,将反应在4℃下孵育过夜 ?转化子的最大数目。

      更详细的信息可以参考此处的PDF协议文件。

  6. 转换
    1. 将5μl连接产物加入50μl感受态大肠杆菌-DH5α中,在冰上孵育30分钟。
    2. 在42℃孵育90秒,然后立即在冰上转移3分钟
    3. 加入1ml LB培养基,在37℃振荡器孵育1小时
    4. 在室温下以8,000rpm离心2分钟并扔掉 ?上清液,用50μlLB培养基重悬细菌 沉淀
    5. 加入2μlIPTG(储存浓度:20%,m/V)和 20μlX-gal(储存浓度:2%,m/V),然后通过移液混合 将溶液铺在Amp电阻板(终浓度Amp: ?50μg/ml)
    6. 将板在37°C细菌培养箱中12-16小时
    7. 选择白色克隆获得HBV DNA整合质粒。

  7. 从 .col i -DH5α提取HBV DNA整合质粒
    1. 从板中分离白色单菌落(步骤F6),并接种a 培养含有Amp抗生素的5ml LB培养基。孵化为 ?12-16小时,在37℃伴随剧烈摇动(?300rpm)。使用10-20毫升 培养管或具有体积至少4倍体积的烧瓶 文化。
    2. 在室温下以10,000×g离心1分钟。
    3. 倾析或吸出并丢弃培养基。
    4. 加入250μlSolution I/RNase A.涡旋或用移液器上下混合 彻底。细胞沉淀的完全重悬对于获得是至关重要的 良好的产量 注意:使用前必须将RNase A添加到Solution I中。请参阅第6页上的准备试剂部分中的说明(附上此处)。
    5. 将悬浮液转移到新的1.5 ml微量离心管中
    6. 加入250μl溶液II。倒转并轻轻旋转几个管 次以获得澄清的裂解物。可能需要2-3分钟的孵育 注意:避免剧烈混合,因为这将剪切染色体DNA和 降低质粒纯度。不要让裂解反应进行得更多 超过5分钟。储存溶液II在不使用时密封,避免 在空气中从CO
    7. 加入350μl溶液III。立即倒置几次,直到形成絮状白色沉淀。
      注意:彻底和立即混合溶液是至关重要的 ?添加溶液III后避免局部沉淀。
    8. 以最大速度(≥13,000×g/min)离心10分钟。将形成紧凑的白色沉淀。立即进行下一步。
    9. 将HiBind ? DNA Mini Column插入2ml收集管。
    10. 通过仔细转移步骤G8的澄清的上清液 将其吸入HiBind DNA Mini Column。小心不要 干扰沉淀,没有细胞碎片转移到 HiBind ? DNA Mini Column。
    11. 以最大速度离心1分钟。
    12. 弃去滤液并重新使用收集管。
    13. 加入500μlHB缓冲液
    14. 以最大速度离心1分钟。
    15. 丢弃滤液和重复使用收集管。
    16. 加入700μlDNA洗涤缓冲液 注意:DNA洗涤缓冲液必须在使用前用100%乙醇稀释。请参阅第6页(附加此处 )以获取说明。
    17. 以最大速度离心1分钟。
    18. 弃去滤液并重新使用收集管。
    19. 将空的HiBind ? DNA Mini Column离心2分钟,以最大速度干燥柱基。
      注意:干燥HiBind ? DNA Mini Column矩阵很重要 洗脱。残余乙醇可能会干扰下游应用。
    20. 将HiBind ? DNA Mini Column转移到干净的1.5 ml微量离心管中。
    21. 加入30-100μl洗脱缓冲液或无菌去离子水直接到柱膜中心 注意:从HiBind DNA Mini Column中洗脱DNA的效率是 ?取决于pH。如果使用无菌去离子水,确保 pH约为8.5。
    22. 让在室温下放置1分钟。
    23. 以最大速度离心1分钟。
      注意:这代表约70%的结合DNA。可选 第二次洗脱将产生任何残留的DNA,虽然在较低 集中。
    24. 质粒测序,选择正确的HBV DNA整合质粒进行下面的步骤
      更详细的信息可以参考此处的PDF协议文件。

  8. 全长HBV基因组整合到克隆载体pUC19
    1. 用 I和 Spe I消化2.05kb-HBV DNA整合的质粒, 1.15-HBV DNA整合质粒与Sac I和Spe I I,具有Sal I和Sac I的pUC19质粒。详细的反应体系如下所述
      试剂
      2.05 kb-HBV DNA-integrated plasmid
      1.15 kb-HBV DNA-整合的质粒
      pUC19
      10x摘要缓冲区
      2微升
      2微升
      2微升
      Sal I
      1微升
      -
      1μl

      1微升
      1微升
      -
      I
      -
      1微升
      1微升
      质粒:
      1微克
      1微克
      1微克
      去离子水至最终体积为
      20微升
      20微升
      20微升

    2. 在37℃孵育1小时
    3. 向每个样品加入10x DNA上样缓冲液,通过移液混合。凝胶 通过在100V下在1%琼脂糖凝胶上电泳30分钟纯化产物
    4. 如步骤C中所述凝胶提取2.05kb片段,1.15kb片段和?2.7kb线性pUC19片段。
    5. 使用T4 DNA连接酶将它们在4℃下整合过夜。详细的反应体系如下所述
      试剂

      10x连接缓冲液
      1微升
      ?2.7kb线性pUC19片段 1微升
      2.05 kb片段
      3微升
      1.15 kb片段
      4微升
      T4 DNA连接酶 1微升
      最终体积为
      10微升

    6. 在4℃下将反应孵育过夜以获得最大数目的转化体。
    7. 按照步骤F1-6中所述将连接产物转化到感受态大肠杆菌-DH5α中。
    8. 按照步骤G从大肠杆菌 i -DH5α提取重组质粒。
    9. 使用 I和 I确定质粒。详细步骤可参考H1-3。
    10. 这三个片段的质粒:2.7kb,2.05kb和1.15kb 通过用Sac I和 Spe I(图2C)消化形成的是正确的 全长HBV基因组整合质粒,pUC/HBV质粒。

代表数据


图1. HBV DNA克隆的示意图。从患者血清中提取HBV DNA作为PCR模板。引物P1和P3用于扩增2.05kb-HBV DNA片段;引物P2和P4用于扩增1.15kb-HBV DNA片段。将两个片段分别连接到pGEM -T载体中,并进行序列分析。从正确的重组质粒中消除了2.05kb-HBV DNA片段和1.15kb-HBV DNA片段,并连接到pUC19质粒中以形成重组pUC/HBV质粒。


图2. pUC/HBV的构建和鉴定 A. 1.15kb HBV DNA片段的扩增。泳道1:标记;泳道2,3:样品。 B.扩增2.05kb HBV DNA片段。泳道1:标记;泳道2,3:样品。 C.通过 Sac I和 Spe I消化鉴定pUC/HBV。泳道1:泳道2:阴性克隆;泳道3:阳性克隆。

食谱

  1. 病毒裂解缓冲液
    20mM Tris-HCl(pH8.0) 10 mM EDTA
    0.1%SDS
    0.8mg/ml蛋白酶K
  2. LB培养基
    10g/L NaCl
    10 g/L胰蛋白酶
    5g/L酵母提取物

致谢

这个协议从以前的工作修改了许石和梁曹。本研究得到中国国家自然科学基金(31200699)的支持。

参考文献

  1. Alcantara,F.F.,Tang,H。和McLachlan,A。(2002)。 乙型肝炎病毒基因组的增强子1区域中干扰素调节元件的功能表征。 a> Nucleic Acids Res 30(9):2068-2075
  2. Cao,L.,Wu,C.,Shi,H.,Gong,Z.,Zhang,E.,Wang,H.,Zhao,K.,Liu,S.,Li,S.,Gao, Wang,Y.,Pei,R.,Lu,M.和Chen,X.(2014)。 乙型肝炎病毒准种的共存增强病毒复制和诱导宿主抗体和细胞免疫应答的能力。 J Virol 88(15):8656-8666。
  3. Gunther,S.,Li,B.C.,Miska,S.,Kruger,D.H.,Meisel,H。和Will,H。(1995)。 有效扩增完整乙型肝炎病毒基因组的新方法允许快速功能分析并揭示缺失突变体免疫抑制的患者。病毒学 69(9):5437-5444。
  4. He,J.,He,L.and Yao,X。(1998)。 研究乙型肝炎病毒全长基因组的新方法。 Chin J Infect Dis 16(2):p70-72。
  5. Huang,L.R.,Wu,H.L.,Chen,P.J.and Chen,D.S。(2006)。 针对人类慢性乙型肝炎病毒感染耐受性的免疫活性小鼠模型。 Proc Natl Acad Sci USA 103(47):17862-17867。
  6. Li,L.,Shen,H.,Li,A.,Zhang,Z.,Wang,B.,Wang,J.,Zheng,X.,Wu,J.,Yang,D.,Lu, Song,J。(2013)。 在流体动力注射小鼠中抑制乙型肝炎病毒(HBV)基因表达和HBx基因沉默的复制模型与HBV基因型B的新克隆。 病毒 10:214.
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cao, L., Wu, C., Zhao, K. and Chen, X. (2015). Characterization of HBV Isolates from Patient Serum Samples and Cloning. Bio-protocol 5(24): e1683. DOI: 10.21769/BioProtoc.1683.
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Health Clinic
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