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Introduction and Sequencing of Patient-isolated HBV RT Sequences into the HBV 1.2-mer Replicon
把亲体分离的HBV RT序列引入HBV 1.2-mer复制子中并测序   

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本实验方案简略版
Journal of Virology
Jun 2014

 

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), Drug resistance (耐药性), Replication (复制), Reverse transcription (反转录)

Materials and Reagents

  1. Sera of patients with chronic hepatitis B (100~200 μl)
  2. QIAamp MinElute virus spin kit (QIAGEN, catalog number: 57704 )
  3. Ex taq polymerase (Takara Bio Company, catalog number: RR001A )
  4. Forward primer (XhoI site is underlined): 5’-AAT CTT CTC GAG GAC TGG GGA CCC TGC ACC-3’
  5. Reverse primer (NcoI site is underlined): 5’-GAG CAG CCA TGG GAA GGA GGT GTA TTT CCG-3’
  6. Gel/PCR DNA Extraction kit (Real Biotech Corporation, catalog number: YDF100 )
  7. pGEM-T Easy vector (Promega Corporation, catalog number: A1360 )
  8. T4 ligase (Takara Bio Company, catalog number: 2011A )
  9. LB broth high salt (Duchefa Biochemie, catalog number: L1704 )
  10. MacConkey agar (BD, catalog number: 212123 )
  11. LaboPass Plasmid Mini Purification Kit (COSMO Genetech, catalog number: CMP0111 )
  12. HBV WT complete sequence (genotype C, GQ872210)
  13. Xho I (New England BioLabs, catalog number: R0146S )
  14. Nco I (New England BioLabs, catalog number: R0193S )
  15. CutSmart™ buffer (New England BioLabs, catalog number: B7204S )
  16. HBV 1.2 replicon (Figure 1)
  17. TE buffer (see Recipes)


    Figure 1. A plasmid map of the HBV 1.2-mer replicon with XhoI and NcoI sites highlighted

Equipment

  1. Centrifuge (Eppendorf, catalog number: 5415R )
  2. Pipet Aid XP (Drummond Scientific Company, catalog number: 4-000-202-E )
  3. MJ Mini 48-Well Personal Thermal Cycler (Bio-Rad Laboratories, catalog number: PTC-1148 )
  4. Nanodrop Spectrophotometer (Nanodrop, catalog number: ND-1000 )
  5. Heat block

Procedure

  1. Amplification of HBV RT domain from patients’ sera
    1. Prepare 100~200 μl of patient’ serum.
    2. Extract the HBV DNA using QIAamp MinElute virus spin kit according to the manufacturer’s protocol and elute viral DNA in 50 μl of elution buffer (provided in the kit) or distilled water.
    3. Amplify HBV RT domain with primers following composition.
      HBV DNA (50 ng/μl)
      1 μl
      Forward primer (10 pM)
      1 μl
      Reverse primer (10 pM)
      1 μl
      10x Ex Taq polymerase buffer
      2 μl
      dNTP mixture (2.5 mM)
      2 μl
      Ex Taq polymerase (5 U/μl )
      0.2μl
      Distilled water
      12.8 μl
      Total
      20 μl
    4. Run PCR as follows: 95 °C for 5 min, followed by 30 cycles of 95 °C for 50 sec, 62 °C for 50 sec, 72 °C 1 min 20 sec, and final extensions at 72 °C for 10 min.
    5. Identify the PCR products (HBV RT domain) on 1% agarose gel by electrophoresis (Figure 2) and purify with Gel/PCR DNA Extraction kit according to the manufacturer’s protocol (final elution volume is 30 μl of TE buffer).

  2. Sequencing of HBV RT domain
    1. Mix the ligation reactions in 1.5 ml tube as described below and incubate overnight at 4 °C.
      Purified PCR products (60 ng/μl )
      1 μl
      pGEM-T Easy vector (50 ng/μl )
      5 μl
      2x Rapid Ligation buffer
      0.3 μl
      T4 DNA ligase (3 Weiss units/μl )
      2.7 μl
      Distilled water
      1 μl
      Total
      10 μl
    2. To inactivate the T4 ligase, incubate the ligation reactions at 65 °C for 15 min and place in ice.
    3. During step B2, prepare the E.Coli (DH5α) competent cells from -80 °C storage and place in ice until thawed.
    4. Transfer 50 μl of competent cells into ligation reactions and incubate in ice for 15 min.
    5. Heat-shock the competent cells for 90 sec in heat block at 42 °C, then immediately incubate in ice for 15 min.
    6. Add 500 μl LB medium to tube from step B5 and incubate for 2 h at 37 °C with shaking.
    7. Centrifuge the tube at 3,000 rpm for 5 min at room temperate, and then remove the 350 μl of supernatant.
    8. Gently mix and plate the residue supernatant (approximate 150 μl) onto Ampr MacConkey agar plates using spreader.
    9. Incubate the plates overnight at 37 °C and check the number of white colonies (approximately 100 colonies should be observed).
    10. Pick the white colonies, then transfer into 2 ml of LB medium with ampicillin in 15 ml tube and incubate overnight at 37 °C with shaking.
    11. Extract the DNA plasmids contained the HBV RT domain from bacterial culture (step B10) using the LaboPass Plasmid Mini Purification Kit following the manufacturer’s protocol and elutes plasmids in 50 μl of EB buffer (provided in Kit) or TE buffer.
    12. Confirm the correct insert DNA by simultaneous digestion reactions for 2 h at 37 °C as described below.
      Plasmid DNA (200 ng)
      -
      XhoI (20 U/μl )
      0.25 μl
      NcoI (10 U/μl )
      0.25 μl
      10x CutSmartTM Buffer
      1 μl
      Distilled water
      up to 10 μl
      Total
      10 μl
    13. Sequencing the HBV RT domain by T7 (5’-TAA TAC GAC TCA CTA TAG GG-3’) and SP6 (5’- ATT TAG GTG ACA CTA TAG-3’) promoter primers (located in pGEM-T Easy vector) and analyze the mutations compared with WT HBV (genotype C, GQ872210).

  3. Cloning of HBV RT domain into HBV 1.2 replicon
    1. Simultaneously digest 200 ng of plasmids [HBV RT plasmid (step B12) for insert DNA and HBV 1.2mer replicon for vector DNA] with digestion reactions (see the procedure C step 12).
    2. Identify 1.2 kb (insert DNA, step B12) and 4.8 kb (vector DNA) fragment on 0.8% agarose gel by electrophoresis (Figure 3) and purify the digested DNAs with Gel/PCR DNA Extraction kit according to the manufacturer’s protocol (final elution volume is 30 μl of TE buffer).
    3. Mix the ligation reactions in 1.5 ml tube as described below and incubate overnight at 4 °C.
      Insert DNA (40 ng/μl)
      1 μl
      Vector DNA (50 ng/μl)
      1 μl
      10x T4 ligase buffer
      1 μl
      T4 DNA ligase (3 Weiss units/μl)
      0.3 μl
      Distilled water
      6.7 μl
      Total
      10 μl
    4. Transform (see steps B2~7) and plate onto Ampr LB agar plates.
    5. Isolate and confirm the plasmid DNA (see steps B9~13).

Representative data



Figure 2. Representative data of amplified HBV RT gene. Prepared HBV DNA from patients sera were used as template. The product size is approximately 1.2 kb.


Figure 3. Confirmation of HBV 1.2-mer replicon. Completed HBV 1.2mer replicon was digested with XhoI, NcoI restriction enzyme and was separated on a 1% agarose gel.

Recipes

  1. TE buffer
    10 mM Tris (pH 8.0)
    1 mM EDTA

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. 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.
  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.

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, 耐药性, 复制, 反转录

材料和试剂

  1. 慢性乙型肝炎患者血清(100〜200μl)
  2. QIAamp MinElute病毒旋转试剂盒(QIAGEN,目录号:57704)
  3. Ex taq聚合酶(Takara Bio Company,目录号:RR001A)
  4. 正向引物( XhoI 位点加下划线):5'-AAT CTT CTC GAG GAC TGG GGA CCC TGC ACC-3'
  5. 反向引物( NcoI 位点加下划线):5'-GAG CAG CCA TGG GAA GGA GGT GTA TTT CCG-3'
  6. Gel/PCR DNA提取试剂盒(Real Biotech Corporation,目录号:YDF100)
  7. pGEM-T Easy载体(Promega Corporation,目录号:A1360)
  8. T4连接酶(Takara Bio Company,目录号:2011A)
  9. LB肉汤高盐(Duchefa Biochemie,目录号:L1704)
  10. MacConkey琼脂(BD,目录号:212123)
  11. LaboPass Plasmid Mini Purification Kit(COSMO Genetech,目录号:CMP0111)
  12. HBV WT完整序列(基因型C,GQ872210)
  13. Xho I (New England BioLabs,目录号:R0146S)
  14. Nco I (New England BioLabs,目录号:R0193S)
  15. CutSmart TM缓冲液(New England BioLabs,目录号:B7204S)
  16. HBV 1.2复制子(图1)
  17. TE缓冲区(参见配方)


    图1. 突出显示XhoI和NcoI位点的HBV 1.2聚体复制子的质粒图

设备

  1. 离心机(Eppendorf,目录号:5415R)
  2. Pipet Aid XP(Drummond Scientific Company,目录号:4-000-202-E)
  3. MJ Mini 48孔个人热循环仪(Bio-Rad Laboratories,目录号:PTC-1148)
  4. Nanodrop分光光度计(Nanodrop,目录号:ND-1000)
  5. 热块

程序

  1. 从患者血清中扩增HBV RT结构域
    1. 准备100〜200μl的患者血清
    2. 使用提取HBV DNA QIAamp MinElute病毒旋转试剂盒根据制造商的方案 并在50μl洗脱缓冲液(试剂盒中提供)或洗脱病毒DNA 蒸馏水
    3. 用引物组成后扩增HBV RT结构域
      HBV DNA(50ng /μl)
      1微升
      正向引物(10pM)
      1微升
      反向引物(10 pM)
      1微升
      10x Ex Taq聚合酶缓冲液
      2微升
      dNTP混合物(2.5mM) 2微升
      Ex Taq聚合酶(5U /μl)
      0.2μl
      蒸馏水
      12.8微升
      总计
      20微升
    4. 运行PCR如下:95℃5分钟,然后是95℃的30个循环 50秒,62℃50秒,72℃1分钟20秒,和最终延伸 在72℃下10分钟
    5. 确定PCR产物(HBV RT结构域)   1%琼脂糖凝胶电泳(图2),并用Gel/PCR纯化 DNA提取试剂盒根据制造商的方案(最终 洗脱体积为30μlTE缓冲液)。

  2. HBV RT结构域的测序
    1. 如下所述在1.5ml管中混合连接反应物,并在4℃下孵育过夜
      纯化的PCR产物(60ng /μl)
      1微升
      pGEM-T Easy载体(50ng /μl)
      5微升
      2x快速连接缓冲液
      0.3微升
      T4 DNA连接酶(3 Weiss单位/μl) 2.7微升
      蒸馏水
      1微升
      总计
      10微升
    2. 为了失活T4连接酶,将连接反应物在65℃下孵育15分钟并置于冰中
    3. 在步骤B2期间,从-80℃储存制备E.Coli (DH5α)感受态细胞,并置于冰中直至解冻。
    4. 转移50微升感受态细胞连接反应,在冰中孵育15分钟
    5. 在42℃的热块中热休克感受态细胞90秒,然后立即在冰中孵育15分钟。
    6. 从步骤B5加入500μlLB培养基,并在37℃下振荡孵育2小时
    7. 在室温下以3,000rpm离心管5分钟,然后取出350μl上清液
    8. 使用扩散器轻轻混合并将残余物上清液(约150μl)平板接种到AmpMacConkey琼脂平板上。
    9. 孵育板在37℃过夜,并检查白色的数量   菌落(应观察到约100个菌落)
    10. 挑 白色菌落,然后转移到2ml含有氨苄青霉素的LB培养基中 在15ml管中并在37℃下振荡孵育过夜
    11. 从细菌中提取含有HBV RT结构域的DNA质粒 使用LaboPass Plasmid Mini Purification Kit进行培养(步骤B10) 按照制造商的方案并洗脱质粒在50微升EB   缓冲液(Kit中提供)或TE缓冲液
    12. 通过如下所述在37℃下同时消化反应2小时来确认正确的插入DNA
      质粒DNA(200ng)
      -
      XhoI (20 U /μl)
      0.25μl
      NcoI (10 U /μl)
      0.25μl
      10x CutSmart TM 缓冲区
      1微升
      蒸馏水
      最多10μl
      总计
      10微升
    13. 通过T7(5'-TAA TAC GAC TCA CTA TAG)对HBV RT结构域进行测序 GG-3')和SP6(5'-ATT TAG GTG ACA CTA TAG-3')启动子引物 (位于pGEM-T Easy载体)并分析突变比较 WT HBV(基因型C,GQ872210)。

  3. 将HBV RT结构域克隆入HBV 1.2复制子
    1. 同时消化200ng质粒[HBV RT质粒(步骤B12) 插入DNA和HBV 1.2mer复制子用于载体DNA] 反应(参见方法C步骤12)。
    2. 识别1.2 kb(插入   DNA,步骤B12)和在0.8%琼脂糖凝胶上的4.8kb(载体DNA)片段 电泳(图3),并用Gel/PCR DNA纯化消化的DNA   根据制造商的方案提取提取试剂盒(最终洗脱 体积为30μlTE缓冲液)
    3. 如下所述在1.5ml管中混合连接反应物,并在4℃下孵育过夜
      插入DNA(40 ng /μl)
      1微升
      载体DNA(50ng /μl)
      1微升
      10×T4连接酶缓冲液
      1微升
      T4 DNA连接酶(3 Weiss单位/μl) 0.3微升
      蒸馏水
      6.7微升
      总计
      10微升
    4. 转化(见步骤B2〜7),并平板接种到Amp LB琼脂平板上
    5. 分离并确认质粒DNA(参见步骤B9〜13)。

代表数据



图2.扩增的HBV RT基因的代表性数据。将来自患者血清的制备的HBV DNA用作模板。 产品尺寸约为1.2 kb。


图3. HBV 1.2-mer复制子的确认。完整的HBV 1.2mer复制子用XhoI,NcoI限制酶消化,并在1%琼脂糖凝胶上分离。

食谱

  1. TE缓冲区
    10mM Tris(pH8.0) 1mM EDTA

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重叠变化的影响 在表面基因中。 J Virol 88(12):6805-6818。
  2. 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) 。 从慢性乙型肝炎患者分离的乙型肝炎病毒的clevudine抗性突变体的鉴定和表征 J Virol 84(9):4494-4503。
  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。
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引用: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. DOI: 10.21769/BioProtoc.1449.
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