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RNA Strand Displacement Assay for Hepatitis E Virus Helicase
戊型肝炎病毒解旋酶的RNA链置换测定   

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Abstract

The hepatitis E virus (HEV) helicase uses ATP to unwind the RNA duplexes. This is an essential step for viral replication. This protocol aims to measure the double strand RNA unwinding activity of the HEV helicase.

Keywords: Hepatitis E virus(戊型肝炎病毒), RNA virus(RNA病毒), Helicase(解旋酶), Helicase assay(解旋酶测定), Viral replication(病毒复制), Nonradioactive helicase assay(非放射性解旋酶测定)

Background

The RNA unwinding activity of HEV helicase has been measured using radiolabeled double stranded RNA (dsRNA, Karpe et al., 2010). We have established a non-radioactive assay protocol for measuring the dsRNA unwinding activity of the HEV helicase. This assay utilizes a fluorescently tagged RNA to measure the activity of the HEV helicase protein purified from human hepatoma cell, thus eliminating the need to handle radioactive material.

Materials and Reagents

  1. 1.5 ml microcentrifuge tube
  2. 60 mm plates
  3. 1.6 ml RNase free microcentrifuge tube
  4. PVDF (Polyvinylidene fluoride) membrane (Pall, catalog number: BSP0149 )
  5. Mammalian expression plasmids (details in Nair et al., 2016):
    1. pCDNA5 Helicase-flag [HEV helicase sequence was PCR amplified using primers: pCDNA5 HEL-flag FP, pCDNA5 HEL-flag RP; digested with HindIII and ligated into pCDNA5 vector digested with HindIII and EcoRV]
    2. pUNO ORF2-flag [ORF2 with C-terminal Flag tag was PCR amplified with primers: pUNO ORF2-flag FP, pUNO ORF2-flag RP; digested with BglII and ligated into pUNO vector digested with NheI [blunted] and BamHI]
  6. Huh7 human hepatoma cells (obtained from Dr. C. M. Rice; Blight et al., 2000)
  7. RNA oligos: HRNA1 5’-UUUUUUUUUUUUCGCUGAUGUCGCCUGG-3’ and HRNA2 5’-[6FAM] CCAGGCGACAUCAGCG-3’ (Sigma-Aldrich, USA)
  8. Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9937 )
  9. Ethidium bromide solution (Sigma-Aldrich, catalog number: E1510 )
  10. Glycogen (Sigma-Aldrich, catalog number: G0885 )
  11. Ethanol (EMD Millipore, catalog number: 100983 )
  12. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  13. DMEM
  14. 10% fetal bovine serum (FBS)
  15. Phosphate-buffered saline (PBS) (Bio Basic, catalog number: PD0100 )
  16. Flag M2 agarose resin (Sigma-Aldrich, catalog number: A2220 )
  17. Flag peptide (Sigma-Aldrich, catalog number: F4799 )
  18. DEPC-treated water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9922 )
  19. Octa-probe antibody (Santa Cruz Biotechnology, catalog number: sc-807 )
  20. Skimmed milk (Sigma-Aldrich, catalog number: 70166 )
    Note: This product has been discontinued.
  21. Anti-rabbit IgG Horseradish peroxidase(HRPO) (Santa Cruz Biotechnology, catalog number: sc-2004 )
  22. Clarity Western ECL blotting substrate (Bio-Rad Laboratories, catalog number: 1705061 )
  23. Silver stain kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 24612 )
  24. Bradford assay reagent (Bio-Rad Laboratories, catalog number: 5000002 )
  25. RNAsin
  26. ATP
  27. Proteinase K (Sigma-Aldrich, catalog number: P2308 )
  28. Glycogen (Sigma-Aldrich, catalog number: G0885 )
  29. Tris (Sigma-Aldrich, catalog number: T6066 )
  30. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  31. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  32. Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
  33. SDS (Sigma-Aldrich, catalog number: L3771 )
  34. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’,-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: 03777 )
  35. Triton X-100 (Sigma-Aldrich, catalog number: 93418 )
    Note: This product has been discontinued.
  36. Sodium pyrophosphate tetrabasic decahydrate (Na4P2O4·10H2O) (Sigma-Aldrich, catalog number: S6422 )
  37. β-glycerol phosphate (Sigma-Aldrich, catalog number: 50020 )
  38. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: S6508 )
  39. Protease inhibitor cocktail tablet (Roche Diagnostics, catalog number: 04693132001 )
  40. HEPES (Sigma-Aldrich, catalog number: H8651 )
  41. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  42. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  43. BSA
  44. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  45. Tween 20
  46. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  47. Xylene cyanol FF (Sigma-Aldrich, catalog number: X4126 )
  48. Annealing buffer (see Recipes)
  49. Soaking buffer (see Recipes)
  50. IP buffer (see Recipes) 
  51. Strand displacement buffer (see Recipes)
  52. 5x termination buffer (see Recipes)
  53. 2x proteinase K buffer (see Recipes)
  54. Phosphate buffered saline with 0.1% Tween 20 (PBST) (see Recipes)
  55. 50 mg/ml proteinase K (see Recipes)
  56. 10 mg/ml glycogen (see Recipes)
  57. RNA loading dye (see Recipes)

Equipment

  1. Vertical electrophoresis mini apparatus
  2. Gel documentation system (Bio-Rad Laboratories, model: ChemiDocTM MP )
  3. NanoDrop (Thermo Fischer Scientific, USA)
  4. Heat block
  5. Water bath
  6. Refrigerated table top centrifuge
  7. -80 °C deep freezer
  8. Flip flop rocker
  9. 5% CO2 incubator

Procedure

  1. Preparation of fluorescently labeled dsRNA
    1. All steps should be performed using nuclease free reagents.
    2. Resuspend the RNA oligos to 100 pmol/μl in nuclease free water. Resolve 1 μl on 20% non-denaturing PAGE (Polyacrylamide gel electrophoresis), stain with ethidium bromide (2 μl of 10 mg/ml stock in 50 ml running buffer) for 15 min at room temperature and visualize the signal using gel documentation system under UV set.
      Note: This step is necessary to rule out occasional degradation of the oligo from the source itself.
    3. Mix 100 pmole (100 pmole/μl stock) of each oligo in 40 μl annealing buffer, incubate at 95 °C for 30 sec and cool gradually to room temperature.
    4. Resolve the annealed oligos in 20% non-denaturing PAGE and excise the band corresponding to the RNA duplex.
    5. Transfer the excised bands to a fresh 1.5 ml microcentrifuge tube, mince and soak in 500 µl soaking buffer overnight (16 h) in a flip flop rocker at room temperature.
    6. Next day, centrifuge the tubes at 13,000 x g for 5 min at room temperature.
    7. Collect the supernatant into a fresh microcentrifuge tube, add 5 μg glycogen (5 mg/ml stock in water) and 1 ml ice cold absolute ethanol and incubate at -80 °C for 45 min.
    8. Centrifuge at 14,000 x g for 30 min at 4 °C.
    9. Wash the pellet with 500 μl 75% ethanol, centrifuge at 14,000 x g for 10 min at 4 °C.
    10. Remove 75% ethanol and dry the pellet at room temperature.
      Note: Be careful while removing the 75% ethanol, pellet may be lost.
    11. Dissolve the RNA pellet in 30 µl nuclease-free water and incubate at 55 °C for 10 min.
    12. Run 1 μl dsRNA in 20% non-denaturing PAGE. Visualize the fluorescent RNA using the gel documentation system using appropriate setting (6-FAM fluorescent dye setting, excitation: 492 nm, emission: 518 nm).
    13. Quantitate the dsRNA by spectrophotometry using NanoDrop and store in aliquots in RNase free microcentrifuge tubes at -80 °C.

  2. Preparation of flag-affinity purified proteins
    1. Transfect 3 μg each of pUNO RdRp-flag and pUNO ORF2-flag plasmids into ten 60 mm plates (each plasmid transfected into 10 plates, not cotransfected), containing Huh7 human hepatoma cells, using Lipofectamine 2000 in 1:1 ratio, following manufacturer’s instructions. Incubate cells at 37 °C in 5% CO2 incubator. 24 h post-transfection, replace media with 2 ml DMEM supplemented with 10% fetal bovine serum.
    2. Forty eight hours post transfection, wash the cells with 1x PBS and resuspend in 800 μl IP buffer.
    3. Generate a homogenous suspension by repeated pipetting and vortexing, and incubate overnight (16 h) on ice at 4 °C (cold room or fridge).
    4. Next day, clarify the lysate by centrifugation at 13,000 x g, 4 °C, 10 min and collect the supernatant into a fresh 1.6 ml RNase free microcentrifuge tube.
    5. Add 100 μl of flag agarose beads, incubate on a rocker at 4 °C for 4 h, wash 3 x in 1 ml IP buffer each by centrifuging at 800 x g, 1 min, at 4 °C. Add 200 μl flag peptide (0.2 mg/ml in PBS) and incubate on a rocker for 15 min at 4 °C. Centrifuge at 800 x g, 1 min, at 4 °C and collect the supernatant, which contains the eluted protein.
    6. Check an aliquot of the purified protein by Western blotting using octa-probe antibody and silver staining, as mentioned below.
    7. Resolve the proteins by 10% SDS-PAGE and transfer onto a PVDF (Polyvinylidene fluoride) membrane.
    8. Block the membrane using 5% skimmed milk in 1x PBS for 45 min at room temperature, incubate overnight (16 h) with 1:1,000 octa-probe primary antibody diluted using 5% skimmed milk in 1x PBST at 4 °C, wash 3 x in PBST, incubate with 1:5,000 anti-rabbit IgG HRPO secondary antibody diluted using 5% skimmed milk in 1x PBST at room temperature, followed by detection of the signal by enhanced chemiluminescence using ‘clarity Western ECL blotting substrate’.
    9. Acquire the Images and analyze using a gel documentation system. A single band of approximately 27 kDa should be obtained, as illustrated in Nair et al. (2016).
    10. For silver staining, resolve the proteins in 10% SDS-PAGE.
    11. Proceed for silver staining using silver stain kit, following the manufacturer’s protocol (Thermo Fisher Scientific, USA). A major band of approximately 27 kDa (corresponding to the size of the band obtained in Western) should be obtained, as illustrated in Nair et al. (2016). Few additional bands are also detected above 27 kDa, which does not affect the efficiency of the assay in our experience. If expected bands are obtained in Western and silver staining, estimate the respective protein concentration by serial dilutions by Bradford assay and store the protein in single use aliquots at -80 °C.

  3. RNA strand displacement assay
    1. Incubate 1 pmol dsRNA and 2 µg flag affinity purified HEV helicase protein or ORF2 protein in strand displacement buffer at 37 °C for 2 h. Following order may be followed.
      Water
      24 µl
      10x buffer
      4 µl
      RNAsin
      1 µl
      ATP (10 mM)
      4 µl
      Protein
      5 µl
      dsRNA
      2 µl
      Total volume
      40 µl
    2. Use appropriate negative controls such as dsRNA without protein or dsRNA with an unrelated protein (ORF2).
    3. Terminate the reaction by adding 10 µl 5x termination buffer.
    4. Add 50 µl 2x proteinase K buffer and 1 µl proteinase K (50 mg/ml stock solution in water) and incubate for 10 min at 37 °C.
    5. Add 1 µl glycogen (10 mg/ml stock solution in water) and 1 ml of ice-cold absolute ethanol to the samples; incubate at -80 °C for 45 min.
    6. Centrifuge the samples at 14,000 x g, 15 min, 4 °C.
    7. Wash the pellet with 1 ml 75% ethanol and resuspend in 10 µl nuclease free water.
      Note: Be careful while removing the 75% ethanol, pellet may be lost. Although not necessarily a better alternative, it may be possible to minimize pellet loss by gently pipetting out 75% ethanol instead of directly decanting. Irrespective of whatever handling technique preferred, attention should be given not to disturb the pellet.
    8. Heat denature an equal amount of dsRNA containing sample at 95 °C for 10 min.
    9. Mix the samples with 10 µl RNA loading dye and resolve in 20% non-denaturing PAGE to differentiate the migration between single strand (ss) RNA and dsRNA.
    10. Visualize the fluorescent bands using a gel documentation system.

Data analysis

While performing the assay, appropriate controls should be included in order to interpret the data and rule out non-specific signals. Negative controls should include omission of helicase protein and nucleotides from the reaction mixture. A titration experiment using increasing quantities of helicase protein in the assay should also be performed to rule out non-specific signals. An assay using an unrelated protein instead of helicase will also rule out a possible non-specific signal. A sample assay has been illustrated in Figure 1. The effect of other factors or compounds on helicase activity may be evaluated by adding them to the reaction mixture.


Figure 1. Assay of RNA strand displacement by helicase. dsRNA: double strand RNA, ssRNA: single strand RNA, Heat denaturation: the sample was incubated at 95 °C for 10 min before loading into the gel.

Recipes

  1. Annealing buffer
    10 mM Tris-Cl (pH 7.4)
    100 mM NaCl
    1 mM EDTA
  2. Soaking buffer
    0.5 M ammonium acetate
    10 mM EDTA
    1% SDS
  3. IP buffer
    20 mM Tris-Cl (pH 7.4)
    150 mM NaCl
    1 mM EDTA (pH 8.0)
    1 mM EGTA (pH 8.0)
    1% Triton X-100
    2.5 mM sodium pyrophosphate
    1 mM β-glycerol phosphate
    1 mM sodium orthovanadate
    1x protease inhibitor cocktail (25x stock prepared by dissolving one tablet in 2 ml 1x PBS)
  4. Strand displacement buffer
    50 mM HEPES (pH 7)
    2 mM MgCl2
    10 mM KCl
    0.05 mg BSA/ml
    2 mM DTT
    1 mM ATP
  5. 5x termination buffer
    1% SDS
    100 mM EDTA
  6. 2x proteinase K buffer
    300 mM NaCl
    100 mM Tris-Cl (pH 7.5)
    1% SDS
  7. PBST
    1x phosphate-buffered saline
    0.1% Tween 20
  8. 50 mg/ml proteinase K
    50 mg/ml stock solution prepared in nuclease free water
  9. 10 mg/ml glycogen
    10 mg/ml stock solution prepared in nuclease free water
  10. RNA loading dye
    0.5 mM EDTA
    0.025% bromophenol blue
    0.025% xylene cyanol FF

Acknowledgments

The work was funded by Ramalingaswamy fellowship and THSTI core grant to MS. VN is supported by a grant from the Department of Science and Technology, Government of India. The protocol has been adapted from Karpe et al. (2010).

References

  1. Blight, K. J., Kolykhalov, A. A. and Rice, C. M. (2000). Efficient initiation of HCV RNA replication in cell culture. Science 290(5498): 1972-1974.
  2. Karpe, Y. A. and Lole, S. K. (2010). NTPase and 5’ to 3’ RNA duplex-unwinding activities of the hepatitis E virus helicase domain. J Virol 84(7): 3595-602.
  3. Nair, V. P., Anang, S., Subramani, C., Madhvi, A., Bakshi, K., Srivastava, A., Shalimar, Nayak, B., Ct, R. K. and Surjit, M. (2016). Endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis E virus. PLoS Pathog 12(4): e1005521.

简介

乙型肝炎病毒(HEV)解旋酶使用ATP来解开RNA双链体。 这是病毒复制的重要步骤。 该方案旨在测量HEV解旋酶的双链RNA展开活性。

已经使用放射性标记的双链RNA(dsRNA,Karpe等人,2010)测量了HEV解旋酶的RNA展开活性。 我们已经建立了用于测量HEV解旋酶的dsRNA展开活性的非放射性测定方案。 该测定利用荧光标记的RNA来测量从人肝癌细胞纯化的HEV解旋酶蛋白的活性,从而消除了处理放射性物质的需要。

关键字:戊型肝炎病毒, RNA病毒, 解旋酶, 解旋酶测定, 病毒复制, 非放射性解旋酶测定

材料和试剂

  1. 1.5 ml微量离心管
  2. 60毫米板
  3. 1.6毫升无RNase的微量离心管
  4. PVDF(聚偏二氟乙烯)膜(Pall,目录号:BSP0149)
  5. 哺乳动物表达质粒(Nair等人的详细信息,2016):
    1. pCDNA5 Helicase标志[HEV解旋酶序列使用引物PCR扩增:pCDNA5 HEL-flag FP,pCDNA5 HEL-flag RP;用Hind III消化,并连接到用Hind III和RV RV消化的pCDNA5载体中
    2. 用引物pENO ORF2-flag FP,pUNO ORF2-flag RP扩增pUNO ORF2标记[ORF2与C端标记标签进行PCR扩增;用 Bg II进行消化,并连接到用NheI(平端化和BamHⅠ)消化的pUNO载体中, br />
  6. Huh7人肝癌细胞(获自C.M.Let; Dr. Blight等人,2000)
  7. RNA寡核苷酸:HRNA1 5'-UUUUUUUUUUUUCGCUGAUGUCGCCUGG-3'和HRNA2 5' - [6FAM] CCAGGCGACAUCAGCG-3'(Sigma-Aldrich,USA)
  8. 无核酸酶水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9937)
  9. 溴化乙锭溶液(Sigma-Aldrich,目录号:E1510)
  10. 糖原(Sigma-Aldrich,目录号:G0885)
  11. 乙醇(EMD Millipore,目录号:100983)
  12. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen< sup>,目录号:11668019)
  13. DMEM
  14. 10%胎牛血清(FBS)
  15. 磷酸盐缓冲盐水(PBS)(Bio Basic,目录号:PD0100)
  16. Flag M2琼脂糖树脂(Sigma-Aldrich,目录号:A2220)
  17. 标记肽(Sigma-Aldrich,目录号:F4799)
  18. DEPC处理水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9922)
  19. Octa探针抗体(Santa Cruz Biotechnology,目录号:sc-807)
  20. 脱脂乳(Sigma-Aldrich,目录号:70166)
    注意:本产品已停产。
  21. 抗兔IgG辣根过氧化物酶(HRPO)(Santa Cruz Biotechnology,目录号:sc-2004)
  22. Clarity Western ECL印迹底物(Bio-Rad Laboratories,目录号:1705061)
  23. 银色试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:24612)
  24. Bradford测定试剂(Bio-Rad Laboratories,目录号:5000002)
  25. RNAsin
  26. ATP
  27. 蛋白酶K(Sigma-Aldrich,目录号:P2308)
  28. 糖原(Sigma-Aldrich,目录号:G0885)
  29. Tris(Sigma-Aldrich,目录号:T6066)
  30. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  31. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  32. 乙酸铵(Sigma-Aldrich,目录号:A1542)
  33. SDS(Sigma-Aldrich,目录号:L3771)
  34. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N', - 四乙酸(EGTA)(Sigma-Aldrich,目录号:03777)
  35. Triton X-100(Sigma-Aldrich,目录号:93418)
    注意:本产品已停产。
  36. 焦磷酸钠四水合物(Na 4 P 2 O 4·10H 2 O)(Sigma-Aldrich,目录号号码:S6422)
  37. 磷酸β-甘油酯(Sigma-Aldrich,目录号:50020)
  38. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:S6508)
  39. 蛋白酶抑制剂混合片(Roche Diagnostics,目录号:04693132001)
  40. HEPES(Sigma-Aldrich,目录号:H8651)
  41. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  42. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  43. BSA
  44. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  45. 吐温20
  46. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126)
  47. 二甲苯cyanol FF(Sigma-Aldrich,目录号:X4126)
  48. 退火缓冲液(见配方)
  49. 浸泡缓冲液(见配方)
  50. IP缓冲区(请参阅配方)
  51. 绞线位移缓冲器(见配方)
  52. 5x终止缓冲区(见配方)
  53. 2x蛋白酶K缓冲液(参见食谱)
  54. 含0.1%吐温20(PBST)的磷酸缓冲盐水(见配方)
  55. 50 mg/ml蛋白酶K(参见食谱)
  56. 10 mg/ml糖原(见食谱)
  57. RNA加载染料(见配方)

设备

  1. 垂直电泳迷你装置
  2. 凝胶记录系统(Bio-Rad Laboratories,型号:ChemiDoc TM/MP)
  3. NanoDrop(Thermo Fischer Scientific,USA)
  4. 热块
  5. 水浴
  6. 冷藏台式离心机
  7. -80°C深层冷冻机
  8. 触发器摇杆
  9. 5%CO 2培养箱

程序

  1. 荧光标记的dsRNA的制备
    1. 所有步骤都应使用不含核酸酶的试剂进行。
    2. 在无核酸酶的水中将RNA寡核苷酸重悬于100 pmol /μl。在20%非变性PAGE(聚丙烯酰胺凝胶电泳)上溶解1μl,用溴化乙锭(2μl,10ml运行缓冲液中的10μg/ml储备液)在室温下染色15分钟,并使用凝胶记录系统显示信号UV设置。
      注意:此步骤是必要的,以排除来自源本身的寡核苷酸的偶尔降解。
    3. 在40μl退火缓冲液中混合每个寡核苷酸的100pmole(100pmole /μl原液),在95℃下孵育30秒,逐渐冷却至室温。
    4. 在20%非变性PAGE中解析退火的寡核苷酸并切除对应于RNA双链体的条带。
    5. 将切除的条带转移到新鲜的1.5ml微量离心管中,在室温下在触发器摇杆中将500μl浸泡缓冲液浸泡过夜(16小时)。
    6. 第二天,在室温下以13,000 x g离心管5分钟
    7. 将上清液收集到新鲜的微量离心管中,加入5μg糖原(5mg/ml水溶液)和1ml冰冷的无水乙醇,并在-80℃下孵育45分钟。
    8. 在4℃下以14,000xg离心30分钟。
    9. 用500μl75%乙醇洗涤沉淀,在4℃下以14,000×g离心10分钟。
    10. 去除75%乙醇,并在室温下干燥沉淀 注意:卸下75%乙醇时要小心,颗粒可能会丢失。
    11. 将RNA沉淀溶解在30μl不含核酸酶的水中,并在55℃下孵育10分钟。
    12. 在20%非变性PAGE中运行1μldsRNA。使用适当设置(6-FAM荧光染料设定,激发:492nm,发射:518nm),使用凝胶文献系统可视化荧光RNA。
    13. 使用NanoDrop通过分光光度法定量dsRNA,并以-80°C储存在无RNase的微量离心管中。
  2. 标记亲和纯化蛋白的制备
    1. 按照制造商的说明,使用Lipofectamine 2000以1:1的比例将pugo RdRp标记和pUNO ORF2标记质粒各3μg转染到10个60mm板(每个质粒转染10个平板,不共转染)中,其中包含Huh7人肝癌细胞。在37℃,5%CO 2培养箱中孵育细胞。转染后24小时,用补充有10%胎牛血清的2ml DMEM代替培养基
    2. 转染后48小时,用1×PBS洗涤细胞并重悬于800μlIP缓冲液中
    3. 通过反复移液和涡旋产生均匀的悬浮液,并在4℃冰冷下孵育过夜(16小时)(冷藏室或冰箱)。
    4. 第二天,通过以13,000 x g,4℃,10分钟离心澄清裂解物,并将上清液收集到新鲜的1.6毫升无RNA酶的微量离心管中。
    5. 加入100μl标志琼脂糖珠,在4°C的摇臂上孵育4 h,每次1 ml IP缓冲液中洗3次,每次通过在4℃下以800 x g,1分钟离心。加入200μl标记肽(0.2mg/ml,在PBS中),并在4℃下在摇臂上孵育15分钟。在800℃离心1分钟,4℃离心,收集含有洗脱蛋白质的上清液。
    6. 如下所述,使用八探针抗体和银染,通过Western印迹检查纯化蛋白的等分试样。
    7. 通过10%SDS-PAGE分析蛋白质并转移到PVDF(聚偏二氟乙烯)膜上
    8. 在室温下用1×PBS中的5%脱脂牛奶封闭膜45分钟,用4℃下的1×PBST中的5%脱脂奶稀释的1:1,000八面体探针一抗孵育过夜(16小时),洗涤3次在PBST中,在室温下用1×PBST中的5%脱脂乳稀释的1:5000抗兔IgG HRPO二级抗体进行孵育,然后使用"透明度Western ECL印迹底物"通过增强的化学发光检测信号。
    9. 获取图像并使用凝胶文件系统分析。应该获得约27kDa的单一条带,如Nair等人所示。 (2016)。
    10. 对于银染色,在10%SDS-PAGE中分辨蛋白质
    11. 按照制造商的协议(Thermo Fisher Scientific,USA),使用银色试剂盒进行银染。如Nair等人所示,应该获得大约27kDa的主要带(对应于在西方获得的频带的大小)。 (2016)。在27 kDa以外也检测到另外的条带,这在我们的经验中不影响测定的效率。如果在Western和银染色中获得预期的条带,则通过Bradford测定法通过连续稀释度估计各自的蛋白质浓度,并将蛋白质在-80℃的一次使用等分试样中储存。

  3. RNA链置换分析
    1. 孵育1 pmol dsRNA和2μg标记亲和纯化的HEV解旋酶蛋白或ORF2蛋白在37℃的链置换缓冲液中2小时。可以遵循以下顺序。

      24μl
      10倍缓冲区
      4μl
      RNAsin
      1μl
      ATP(10 mM)
      4μl
      蛋白质
      5μl
      dsRNA
      2μl
      总音量
      40μl
    2. 使用适当的阴性对照,如dsRNA,不含蛋白质或dsRNA与不相关的蛋白质(ORF2)
    3. 通过加入10μl5x终止缓冲液终止反应。
    4. 加入50μl2x蛋白酶K缓冲液和1μl蛋白酶K(50mg/ml储备液在水中),并在37℃下孵育10分钟。
    5. 向样品中加入1μl糖原(10mg/ml水溶液)和1ml冰冷的无水乙醇;在-80℃下孵育45分钟
    6. 以14,000 x g,15分钟,4℃离心样品。
    7. 用1 ml 75%乙醇洗涤沉淀,并重悬于10μl无核酸酶的水中 注意:卸下75%乙醇时要小心,颗粒可能会丢失。虽然不一定是更好的替代方案,但是可以通过轻轻移取75%乙醇而不是直接倾析来最大限度地减少沉淀物的损失。无论采用何种处理技术,首先应注意不要打扰沉淀。
    8. 加热使相同数量的含dsRNA的样品在95℃下变性10分钟
    9. 将样品与10μlRNA加载染料混合,并在20%非变性PAGE中分解,以区分单链(ss)RNA和dsRNA之间的迁移。
    10. 使用凝胶文件系统可视化荧光条带。

数据分析

在进行测定时,应包括适当的控制以解释数据并排除非特异性信号。阴性对照应包括从反应混合物中排除解旋酶蛋白和核苷酸。还应进行在测定中使用越来越多的解旋酶蛋白的滴定实验以排除非特异性信号。使用不相关蛋白而不是解旋酶的测定也将排除可能的非特异性信号。图1中示出了样品测定。其他因素或化合物对解旋酶活性的影响可以通过将它们加入到反应混合物中来评估。


图1.通过解旋酶测定RNA链置换。dsRNA:双链RNA,ssRNA:单链RNA,热变性:将样品在95℃下孵育10分钟,然后加载到凝胶中。

食谱

  1. 退火缓冲器
    10mM Tris-Cl(pH 7.4)
    100mM NaCl
    1 mM EDTA
  2. 浸泡缓冲区
    0.5M醋酸铵
    10 mM EDTA
    1%SDS
  3. IP缓冲区
    20mM Tris-Cl(pH7.4)
    150 mM NaCl
    1mM EDTA(pH8.0)
    1mM EGTA(pH8.0)
    1%Triton X-100
    2.5mM焦磷酸钠
    1mMβ-甘油磷酸盐
    1mM原钒酸钠
    1x蛋白酶抑制剂混合物(通过将一片溶解在2ml 1x PBS中制备的25x储备液)
  4. 股线位移缓冲器
    50 mM HEPES(pH 7)
    2mM MgCl 2
    10 mM KCl
    0.05 mg BSA/ml
    2 mM DTT
    1 mM ATP
  5. 5x终止缓冲区
    1%SDS
    100 mM EDTA
  6. 2x蛋白酶K缓冲液
    300 mM NaCl
    100mM Tris-Cl(pH7.5)
    1%SDS
  7. PBST
    1x磷酸盐缓冲盐水
    0.1%吐温20
  8. 50 mg/ml蛋白酶K
    在无核酸酶的水中制备50 mg/ml储备溶液
  9. 10毫克/毫升糖原
    在无核酸酶水中制备的10mg/ml储备溶液
  10. RNA加载染料
    0.5 mM EDTA
    0.025%溴酚蓝
    0.025%二甲苯cyanol FF

致谢

这项工作由Ramalingaswamy奖学金和THSTI授予MS资助。 VN得到印度政府科学技术部的资助。该协议已经从Karpe等人改编。 (2010)。

参考文献

  1. Blight,KJ,Kolykhalov,AA和Rice,CM(2000)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/11110665"target ="在细胞培养中高效启动HCV RNA复制。 科学 290(5498):1972-1974。
  2. Karpe,YA和Lole,SK(2010)。 NTPase和乙肝病毒解旋酶结构域的5'至3'RNA双链体展开活性。 84(7):3595-602。
  3. Nair,Anang,S.,Subramani,C.,Madhvi,A.,Bakshi,K.,Srivastava,A.,Shalimar,Nayak,B.,Ct,RK和Surjit,M。(2016)。 内质网压力诱导的新型病毒因子的合成介导有效复制基因型1型戊型肝炎病毒。 PLoS Pathog 12(4):e1005521。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nair, V. P. and Surjit, M. (2017). RNA Strand Displacement Assay for Hepatitis E Virus Helicase. Bio-protocol 7(7): e2198. DOI: 10.21769/BioProtoc.2198.
  2. Nair, V. P., Anang, S., Subramani, C., Madhvi, A., Bakshi, K., Srivastava, A., Shalimar, Nayak, B., Ct, R. K. and Surjit, M. (2016). Endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis E virus. PLoS Pathog 12(4): e1005521.
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