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RNA-dependent RNA Polymerase Assay for Hepatitis E Virus
戊型肝炎病毒依赖于RNA的RNA聚合酶活性测定   

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Abstract

RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to assay the RdRp activity of many other viruses, this assay utilizes DIG-11-UTP as a nonradioactive alternative to 32P-UTP, thereby increasing the convenience of performing the assay.

Keywords: Hepatitis E virus(戊型肝炎病毒), RNA virus(RNA病毒), RdRp(RdRp), RdRp assay(RdRp测定), Viral replication(病毒复制), Nonradioactive RdRp assay(非放射性RdRp测定)

Background

No assay was available to measure the activity of HEV RdRp. RdRp activity has been measured in few other viruses such as hepatitis C virus using a radiolabeled nucleotide (Behrens et al., 1996). We have adapted the protocol described by Behrens et al. (1996) and modified it to establish a non-radioactive assay protocol, which is dependent on incorporation of DIG-11-UTP into the antisense RNA strand as a measure of the activity of HEV RdRp. This assay utilizes an in vitro synthesized viral RNA fragment as a template to measure the activity of HEV RdRp protein purified from human hepatoma cells using a chemiluminescence-based strategy.

Materials and Reagents

  1. 60 mm plates
  2. 1.6 ml RNase free microcentrifuge tube
  3. PVDF (Polyvinylidene fluoride) membrane (Pall, catalog number: BSP0149 )
  4. Nylon membrane (Pall, catalog number: 60207 )
  5. Whatman filter paper (Sigma-Aldrich, catalog number: WHA10426972 )
  6. Cling film wrap/plastic food wrap (available in general stores/supermarkets)
  7. Blotting paper (Praveen Scientific, catalog number: PSC 006 )
    Note: It is general blotting paper, used in labs in routine application, similar to but cheaper than Whatman filter paper.
  8. Mammalian expression plasmids (pUNO RdRp-flag [RdRp sequence with C-terminal flag tag was PCR amplified, digested with AgeI, NheI and ligated into pUNO vector digested with same], 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); Nair et al. [2016])
  9. Huh7 (human hepatoma cells, obtained from Dr. C. M. Rice; Blight et al. [2000])
  10. pSKHEV2 RdRp template (pSK HRt) plasmid (Genbank No. AF444002.1, Emerson et al., 2004)
  11. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  12. DMEM
  13. 10% fetal bovine serum (FBS)
  14. Phosphate-buffered saline (PBS) (Bio Basic, catalog number: PD0100 )
  15. Flag M2 agarose resin (Sigma-Aldrich, catalog number: A2220 )
  16. Flag peptide (Sigma-Aldrich, catalog number: F4799 )
  17. Octa-probe antibody (Santa Cruz Biotechnology, catalog number: sc-807 )
  18. Skimmed milk powder (Sigma-Aldrich, catalog number: 70166 )
    Note: This product has been discontinued.
  19. Anti-rabbit IgG Horseradish peroxidase(HRPO) (Santa Cruz Biotechnology, catalog number: sc-2004 )
  20. Clarity Western ECL blotting substrate (Bio-Rad Laboratories, catalog number: 1705061 )
  21. Silver stain kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 24612 )
  22. Bradford assay reagent (Bio-Rad Laboratories, catalog number: 5000002 )
  23. StuI, NheI and Tth111I restriction enzymes
  24. BglII (New England Biolabs, catalog number: R0144L )
  25. PCR purification kit (Agilent Technologies, catalog number: 400771 )
  26. mMessage mMachine T7 kit (Thermo Fisher Scientific, AmbionTM, catalog number: AM1344 )
  27. RNAsin
  28. ATP
  29. CTP
  30. GTP
  31. UTP
  32. DIG-UTP
  33. Actinomycin D (Sigma-Aldrich, catalog number: A1410 )
  34. DMSO
  35. RNase A
  36. Proteinase K (Sigma-Aldrich, catalog number: P2308 )
  37. Glycogen (Sigma-Aldrich, catalog number: G0885 )
  38. Ethanol (EMD Millipore, catalog number: 100983 )
  39. Nuclease free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9937 )
  40. Agarose (Bio Basic, catalog number: AB0014 )
  41. DEPC-treated water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9922 )
  42. 37% formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  43. Formamide (Sigma-Aldrich, catalog number: F9037 )
  44. Gel loading dye
  45. Millennium marker (Thermo Fisher Scientific, AmbionTM, catalog number: AM7151 )
  46. Ethidium bromide (Sigma-Aldrich, catalog number: E1510 )
  47. DIG Northern Starter Kit (Roche Diagnostics, catalog number: 12039672910 )
  48. Tris (Sigma-Aldrich, catalog number: T6066 )
  49. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  50. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  51. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’,-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: 03777 )
  52. Triton X-100 (Sigma-Aldrich, catalog number: 93418 )
    Note: This product has been discontinued.
  53. Sodium pyrophosphate tetrabasic decahydrate (Na4P2O4·10H2O) (Sigma-Aldrich, catalog number: S6422 )
  54. β-glycerol phosphate (Sigma-Aldrich, catalog number: 50020 )
  55. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: S6508 )
  56. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693132001 )
  57. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  58. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  59. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  60. SDS
  61. MOPS (Sigma-Aldrich, catalog number: M5162 )
  62. Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
  63. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
  64. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  65. Tri-sodium citrate dihydrate (Himedia, catalog number: RM1415 )
  66. IP (Immunoprecipitation) buffer (see Recipes)
  67. 5x assay buffer (see Recipes)
  68. 2x proteinase K buffer (see Recipes)
  69. 10x MOPS (pH 7.0) (see Recipes)
  70. 20x SSC buffer (see Recipes)
  71. Maleic acid buffer (pH 7.5) (see Recipes)
  72. 1x blocking solution (see Recipes)
  73. Washing buffer (pH 7.5) (see Recipes)
  74. Detection buffer (pH 9.5) (see Recipes)
  75. Phosphate buffered saline with 0.1% Tween 20 (PBST, see Recipes)

Equipment

  1. 5% CO2 incubator
  2. Centrifuge
  3. Flip-flop rocker
  4. Gel documentation system (Bio-Rad Laboratories, model: ChemiDocTM MP )
  5. Chemical fume hood
  6. Conical flask
  7. Standard horizontal agarose gel electrophoresis apparatus
  8. Rectangular glass tray
  9. Glass plates
  10. Glass jar
  11. UV cross linker (UVP, model: CL-1000 )

Procedure

  1. 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. 48 h post transfection, wash the cells with 1x PBS and resuspend each dish 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. Pool lysates of all ten plates and add 100 μl of flag agarose beads, incubate on 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 55 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). A single band of approximately 55 kDa should be obtained as illustrated in Nair et al. (2016).
    12. 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.

  2. Preparation of template for RdRp assay
    1. Fuse HEV genomic regions 1-130 bases and 6,963-7,204 bases (from 5’-end) to each other by removing 131-6,962 bases from pSKHEV2 plasmid. Briefly, pSKHEV2 plasmid was digested with StuI, NheI and Tth111I restriction enzymes, followed by gel extraction of ~3.5 kb band. This fragment was treated with DNA polymerase I klenow fragment to generate blunt end, followed by self-ligation. Resulting plasmid is named as pSK HRt. Plasmid is available on request. This plasmid has been described in our earlier publication (Nair et al., 2016).
    2. Linearize 10 µg pSK HRt plasmid using 50 U BglII ( R0144L , NEB, buffer 3.1) in 50 µl reaction mix (5 µl 10x buffer, 20 µl DNA, 2.5 µl enzyme and 22.5 µl water). Purify the linearized DNA using PCR purification kit. Use 1 µg linearized DNA for in vitro transcription using mMessage mMachine T7 Kit, following manufacturer’s instruction, to generate a 340 nucleotide long capped RNA, which is to be used as template in RdRp assay to monitor antisense strand synthesis.
    3. Verify the size and integrity of the RNA by formaldehyde agarose gel electrophoresis.
    4. Quantify the RNA by spectrophotometry and store in single use aliquots in RNase free microcentrifuge tubes at -80 °C.

  3. RdRp assay
    1. Assemble the reaction as described below in RNase free tubes.
      Nuclease free water:
      16 µl
      5x buffer
      8 µl
      RNAsin
      1 µl
      ATP (10 mM)
      2 µl
      CTP (10 mM)
      2 µl
      GTP (10 mM)
      2 µl
      UTP (10 mM)
      1.5 µl
      DIG-UTP (10 mM)
      0.5 µl
      Actinomycin D (2 mg/ml stock in DMSO)
      1 µl
      Protein (400 ng/µl)
      5 µl
      RNA template (500 ng/µl)
      1 µl
      Total volume
      40 µl
    2. Incubate at 22 °C for 2 h.
    3. Use appropriate negative controls such as RNA template without protein or RNA template with an unrelated protein (ORF2).
    4. Run one additional sample containing RNA template with RdRp and digest it with RNase A (add 1 μl of 10 mg/ml RNase A to the tube, incubate at 37 °C for 10 min).
    5. 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.
    6. 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.
    7. Centrifuge the samples at 14,000 x g, 15 min, 4 °C.
    8. 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.
    9. Resolve the samples by 1.5% formaldehyde agarose gel electrophoresis in a chemical fume hood.

  4. Formaldehyde agarose gel electrophoresis of RNA samples
    1. For 100 ml, add 0.75 g agarose in 72 ml DEPC-treated water in a conical flask and boil. When the solution has cooled to 55 °C, add 18 ml of 37% formaldehyde and 10 ml 10x MOPS. Cast the gel in gel running apparatus and allow to solidify.
      Note: Do not add formaldehyde and MOPS to very hot agarose solution. Perform the entire procedure in a chemical fume hood.
    2. Run the gel for 30 min prior to loading the samples.
    3. Mix 5 µl RNA, 3 µl 10x MOPS, 6 µl formaldehyde, 15 µl formamide and incubate at 85 °C for 10 min, followed by cooling on ice for 2 min. Add 2.5 µl of freshly prepared gel loading dye and load samples into the gel and run at 3-4 V/cm in 1x MOPS until the lower dye has migrated two third of the distance from the top.
    4. Run RNA molecular weight marker (Millennium marker, Ambion, USA) in parallel to monitor the size of the RdRp assay products. Excise the lane corresponding to the marker and stain with ethidium bromide (2 μl of 10 mg/ml stock in 50 ml running buffer) for 15 min to visualize the markers. Proceed with rest of the gel for transfer to nylon membrane.

  5. Transfer of RNA and detection of signal
    1. Rinse the gel twice in 20x SSC buffer for 15 min each.
    2. Incubate the positively charged nylon membrane in DEPC-treated water until it is completely wet from beneath.
    3. Immerse the membrane in 10x SSC buffer for 5 min.
    4. In a glass tray, add 20x SSC buffer and put a glass plate on a glass jar. Spread a Whatman filter paper wick over the glass plate so that the tips are completely immersed in the buffer.
    5. Place the gel in inverted position. Make a notch on left side of the gel.
    6. Place the membrane on the gel without trapping air bubbles.
    7. Cover the sides of the gel with plastic wrap and remove air bubbles.
    8. Place a Whatman paper cut in the same size of the gel on top of the membrane.
    9. Place a stack of blotting paper cut in the same size of the gel on top of the Whatman paper.
    10. Put a glass plate on the stack and keep a 500 g weight support on top of it.
    11. Allow transfer overnight without disturbance.
    12. Next day, dismantle the transfer assembly and keep the membrane on a filter paper.
    13. Fix the RNA to the membrane by UV crosslinking (120 millijoules/cm2) for 1 min.
    14. Rinse the membrane briefly in DEPC-treated water.
    15. Rinse the membrane in washing buffer for 5 min at room temperature.
    16. Incubate the membrane in 20 ml of 1x blocking solution (see Recipes) for 30 min at room temperature.
      Note: Be careful not to allow the membrane to dry at any point from this step onwards.
    17. Incubate in 10 ml of anti-DIG antibody solution (1:10,000 in 1x blocking solution) for 30 min at room temperature. Antibody comes in the DIG Northern Starter Kit.
    18. Equilibrate in 100 ml detection buffer for 5 min at room temperature.
    19. Place the membrane on a development folder and apply CDP star (part of DIG Northern Starter Kit) and incubate for 5 min in the dark at room temperature.
      Note: Remove all air bubbles.
    20. Acquire chemiluminescent images using a gel documentation system or using an X ray film.

Data analysis

While setting up the RdRp assay, appropriate controls should be included in order to interpret the data and rule out non-specific signal. Must have negative controls such as omission of nucleotides from the reaction mixture and inclusion of RNase A in the reaction mixture. No bands should be obtained in both the cases. A titration experiment using increasing quantities of RdRp protein in the assay should also be performed to rule out non-specific signals. Assay using an unrelated protein instead of RdRp will also rule out the possible non-specific signal. A sample assay with all controls has been illustrated in Figure 1. The effect of other factors or compounds on RdRp activity may be evaluated by adding them to the reaction mixture. In our experience, template itself does not give any false signal, as the assay is dependent on detection of DIG, which is incorporated into the RNA only as DIG-UTP. Free DIG-UTP is removed from the reaction during migration of the sample in agarose gel. Moreover, the labeled RNA migrates at ~800 base pairs size whereas template RNA size is 380 base pairs. Monitoring the size of the band also allows one to be confident of the output of the assay.


Figure 1. Assay of HEV RNA-dependent RNA polymerase activity. NTPs: nucleotide triphosphates.

Recipes

  1. IP buffer (store at 4 °C)
    20 mM Tris (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)
  2. 5x assay buffer (store at -20 °C)
    200 mM Tris (pH 7.5)
    100 mM MgCl2
    200 mM DTT
    200 mM KCl
    100 mM EDTA (pH 8.0)
  3. 2x proteinase K buffer
    300 mM NaCl
    100 mM Tris-Cl (pH 7.5)
    1% SDS
  4. 10x MOPS (pH 7.0)
    200 mM MOPS
    50 mM NaAc
    20 mM EDTA (pH 7)
  5. 20x SSC
    3 M NaCl
    300 mM sodium citrate (pH 7)
  6. Maleic acid buffer (pH 7.5)
    100 mM maleic acid
    150 mM NaCl (pH 7.5)
  7. 1x blocking solution
    Dilute the 10x blocking solution available in the DIG Northern Starter Kit with maleic acid buffer to 1x
    Note: Prepare fresh.
  8. Wash buffer (pH 7.5)
    100 mM maleic acid
    150 mM NaCl
    0.3% Tween 20 (pH 7.5)
  9. Detection buffer (pH 9.5)
    100 mM Tris-Cl
    100 mM NaCl (pH 9.5)
  10. Phosphate buffered saline with 0.1% Tween 20 (PBST)
    1x phosphate-buffered saline
    0.1% Tween 20

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 Behrens et al. (1996).

References

  1. Behrens, S. E., Tomei, L. and De Francesco, R. (1996). Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 15(1): 12-22.
  2. 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.
  3. Emerson, S. U., Nguyen, H., Graff, J., Stephany, D. A., Brockington, A. and Purcell, R. H. (2004). In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. J Virol 78(9): 4838-4846.
  4. 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

RNA依赖性RNA聚合酶(RdRp)对RNA病毒的病毒RNA的复制至关重要。 它合成病毒基因组RNA的互补链,其随后用作模板以产生更多的病毒基因组拷贝。 该测定法测定戊型肝炎病毒(HEV)RdRp的活性。 与可用于测定许多其他病毒的RdRp活性的方案相比,该测定法使用DIG-11-UTP作为32P-UTP的非放射性替代物,从而增加了进行测定的便利性。

没有测定可测量HEV RdRp的活性。 已经使用放射性标记的核苷酸(Behrens等人,1996)在少数其他病毒如丙型肝炎病毒中测量了RdRp活性。 我们已经调整了Behrens等人所描述的协议。 (1996),并将其修饰为建立非放射性测定方案,其依赖于将DIG-11-UTP掺入反义RNA链中作为HEV RdRp的活性的量度。 该测定使用体外合成的病毒RNA片段作为模板,以使用基于化学发光的策略来测量从人肝癌细胞纯化的HEV RdRp蛋白的活性。

关键字:戊型肝炎病毒, RNA病毒, RdRp, RdRp测定, 病毒复制, 非放射性RdRp测定

材料和试剂

  1. 60毫米板
  2. 1.6毫升无RNase的微量离心管
  3. PVDF(聚偏二氟乙烯)膜(Pall,目录号:BSP0149)
  4. 尼龙膜(Pall,目录号:60207)
  5. Whatman滤纸(Sigma-Aldrich,目录号:WHA10426972)
  6. 保鲜膜包装/塑料食品包装(一般商店/超级市场)
  7. 印刷纸(Praveen Scientific,目录号:PSC 006)
    注意:它是一般印迹纸,用于常规应用中的实验室,与Whatman滤纸相似但便宜。
  8. 将哺乳动物表达质粒(pUNO RdRp-flag [具有C末端标记的RdRp序列进行PCR扩增,用"Age","Nhe"I消化并连接到用相同的消化的pUNO载体中],pUNO ORF2标记[ORF2与C-末端标签标签用PCR扩增,用引物pUNO ORF2标记FP,pUNO ORF2标记RP;用BglⅡ消化,并连接到pUNO载体中消化我[blunted]和 Bam HI); Nair等人。 [2016])
  9. Huh7(人肝癌细胞,获自C.M.Sm。; Blight等人,[2000])
  10. pSKHEV2 RdRp模板(pSK HRt)质粒(Genbank No.AF444002.1,Emerson等,2004)
  11. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen< sup>,目录号:11668019)
  12. DMEM
  13. 10%胎牛血清(FBS)
  14. 磷酸盐缓冲盐水(PBS)(Bio Basic,目录号:PD0100)
  15. Flag M2琼脂糖树脂(Sigma-Aldrich,目录号:A2220)
  16. 标记肽(Sigma-Aldrich,目录号:F4799)
  17. Octa探针抗体(Santa Cruz Biotechnology,目录号:sc-807)
  18. 脱脂奶粉(Sigma-Aldrich,目录号:70166)
    注意:本产品已停产。
  19. 抗兔IgG辣根过氧化物酶(HRPO)(Santa Cruz Biotechnology,目录号:sc-2004)
  20. Clarity Western ECL印迹底物(Bio-Rad Laboratories,目录号:1705061)
  21. 银色试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:24612)
  22. Bradford测定试剂(Bio-Rad Laboratories,目录号:5000002)
  23. 我我我和第111号限制酶
  24. Bgl II(New England Biolabs,目录号:R0144L)
  25. PCR纯化试剂盒(Agilent Technologies,目录号:400771)
  26. mMessage mMachine T7试剂盒(Thermo Fisher Scientific,Ambion TM,目录号:AM1344)
  27. RNAsin
  28. ATP
  29. CTP
  30. GTP
  31. UTP
  32. DIG-UTP
  33. 放线菌素D(Sigma-Aldrich,目录号:A1410)
  34. DMSO
  35. RNase A
  36. 蛋白酶K(Sigma-Aldrich,目录号:P2308)
  37. 糖原(Sigma-Aldrich,目录号:G0885)
  38. 乙醇(EMD Millipore,目录号:100983)
  39. 无核酸酶水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9937)
  40. 琼脂糖(Bio Basic,目录号:AB0014)
  41. DEPC处理水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9922)
  42. 37%甲醛(Sigma-Aldrich,目录号:F8775)
  43. 甲酰胺(Sigma-Aldrich,目录号:F9037)
  44. 凝胶加载染料
  45. 千年标记(Thermo Fisher Scientific,Ambion TM ,目录号:AM7151)
  46. 溴化乙锭(Sigma-Aldrich,目录号:E1510)
  47. DIG Northern Starter Kit(Roche Diagnostics,目录号:12039672910)
  48. Tris(Sigma-Aldrich,目录号:T6066)
  49. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  50. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  51. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N', - 四乙酸(EGTA)(Sigma-Aldrich,目录号:03777)
  52. Triton X-100(Sigma-Aldrich,目录号:93418)
    注意:本产品已停产。
  53. 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)
  54. 磷酸β-甘油酯(Sigma-Aldrich,目录号:50020)
  55. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:S6508)
  56. 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693132001)
  57. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  58. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  59. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  60. SDS
  61. MOPS(Sigma-Aldrich,目录号:M5162)
  62. 乙酸钠(Sigma-Aldrich,目录号:S2889)
  63. 马来酸(Sigma-Aldrich,目录号:M0375)
  64. 吐温20(Sigma-Aldrich,目录号:P9416)
  65. 柠檬酸三钠二水合物(Himedia,目录号:RM1415)
  66. IP(免疫沉淀)缓冲液(见配方)
  67. 5x测定缓冲液(参见食谱)
  68. 2x蛋白酶K缓冲液(参见食谱)
  69. 10x MOPS(pH 7.0)(见配方)
  70. 20x SSC缓冲液(见配方)
  71. 马来酸缓冲液(pH 7.5)(参见食谱)
  72. 1x封闭解决方案(请参阅配方)
  73. 洗涤缓冲液(pH 7.5)(参见食谱)
  74. 检测缓冲液(pH 9.5)(见配方)
  75. 含0.1%吐温20的磷酸缓冲盐水(PBST,见食谱)

设备

  1. 5%CO 2培养箱
  2. 离心机
  3. 触发器摇杆
  4. 凝胶记录系统(Bio-Rad Laboratories,型号:ChemiDoc TM/MP)
  5. 化学通风柜
  6. 锥形瓶
  7. 标准水平琼脂糖凝胶电泳仪
  8. 矩形玻璃托盘
  9. 玻璃板
  10. 玻璃瓶
  11. 紫外线交联剂(UVP,型号:CL-1000)

程序

  1. 标记亲和纯化蛋白质的制备
    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1x PBS洗涤细胞,并将每个皿重悬在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. 将所有十个平板的溶液裂解并加入100μl标志琼脂糖珠,在4℃的摇床上孵育4小时,在1ml IP缓冲液中洗涤3次,各自通过800g离心1分钟,在4℃。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。如Nair等人所示,应该获得约55kDa的单一条带。 (2016)。
    10. 对于银染色,在10%SDS-PAGE中分辨蛋白质
    11. 按照制造商的方案(Thermo Fisher Scientific),使用银色试剂盒进行银染。如Nair等人所述,应该获得约55kDa的单一条带。 (2016)。
    12. 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。

  2. 制备RdRp分析模板
    1. 通过从pSKHEV2质粒中除去131-6,962个碱基,从而保护HEV基因组区域1-130个碱基和6,963-7,204个碱基(从5'端)。简言之,将pSKHEV2质粒用"Stu"I,N"I"和"T"111I限制酶消化,然后凝胶提取〜3.5kb的条带。用DNA聚合酶I klenow片段处理该片段以产生平端,随后进行自连接。得到的质粒命名为pSK HRt。可根据要求提供质粒。该质粒已在我们早期的出版物(Nair等人,2016)中有所描述。
    2. 使用50μl反应混合物(5μl10x缓冲液,20μlDNA,2.5μl)将50μgpSK HRt质粒在50μl反应混合物(50μl)中,使用50U Bg II(R0144L,NEB,缓冲液3.1) μl酶和22.5μl水)。使用PCR纯化试剂盒纯化线性化DNA。根据制造商的说明,使用mMessage mMachine T7试剂盒,使用1μg线性化DNA进行体外转录,以产生340个核苷酸长封端的RNA,其用作RdRp测定中的模板以监测反义链合成。
    3. 通过甲醛琼脂糖凝胶电泳验证RNA的大小和完整性
    4. 通过分光光度法对RNA进行定量分析,并在-80°C下将其储存在无RNase的微量离心管中。
  3. RdRp分析
    1. 如下所述在无RNA酶的管中装配反应。
      无核酸酶水:
      16μl
      5x缓冲区
      8μl
      RNAsin
      1μl
      ATP(10 mM)
      2μl
      CTP(10 mM)
      2μl
      GTP(10 mM)
      2μl
      UTP(10 mM)
      1.5μl
      DIG-UTP(10 mM)
      0.5μl
      放线菌素D(2毫克/毫升储存在DMSO中)
      1μl
      蛋白质(400 ng /μl)
      5μl
      RNA模板(500ng /μl)
      1μl
      总音量
      40μl
    2. 在22°C孵育2 h
    3. 使用不含蛋白质或RNA模板的RNA模板,使用不相关蛋白(ORF2)等适当的阴性对照
    4. 运行另外一个含有RdRp RNA模板的样品,并用RNase A消化(加入1μl10 mg/ml Rnase A到管中,37℃孵育10分钟)。
    5. 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。
    6. 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
    7. 以14,000 x g,15分钟,4℃离心样品。
    8. 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。
    9. 通过化学通风橱中的1.5%甲醛琼脂糖凝胶电泳解析样品
  4. RNA样品的甲醛琼脂糖凝胶电泳
    1. 对于100ml,在锥形瓶中加入0.75g琼脂糖在72ml DEPC处理过的水中并煮沸。当溶液冷却至55℃时,加入18ml 37%甲醛和10×10 5 MOPS。将凝胶置于凝胶运行装置中并使其固化。
      注意:不要在非常热的琼脂糖溶液中加入甲醛和MOPS。在化学通风橱中执行整个程序。
    2. 运行凝胶30分钟,然后加载样品。
    3. 混合5μlRNA,3μl10x MOPS,6μl甲醛,15μl甲酰胺,并在85℃下孵育10分钟,然后在冰上冷却2分钟。加入2.5μl新鲜制备的凝胶载色染料,并将样品加载到凝胶中,以1×MOPS的3-4V/cm的速度运行,直到较低的染料迁移离顶部距离的两分之一。
    4. 平行运行RNA分子量标记(Millennium标记,Ambion,USA)以监测RdRp测定产物的大小。消除对应于标记物的泳道,并用溴化乙锭(2μl,10ml运行缓冲液中的10mg/ml储备液)染色15分钟以显现标记物。继续使用其余的凝胶转移到尼龙膜。

  5. 转移RNA和信号检测
    1. 在20x SSC缓冲液中冲洗凝胶两次,每次15分钟
    2. 在DEPC处理的水中孵育带正电的尼龙膜,直到其从下方完全湿透
    3. 将膜浸入10×SSC缓冲液中5分钟
    4. 在玻璃托盘中,加入20x SSC缓冲液,并将玻璃板放在玻璃罐上。将Whatman过滤纸芯涂抹在玻璃板上,使尖端完全浸入缓冲液中。
    5. 将凝胶置于相反的位置。在凝胶左侧做一个缺口。
    6. 将膜放在凝胶上,不会吸入气泡。
    7. 用塑料包裹住凝胶的两侧并清除气泡。
    8. 将与Whatman切纸相同尺寸的凝胶放在膜顶部。
    9. 在Whatman论文的顶部放置一叠在同一大小凝胶上的吸墨纸。
    10. 将玻璃板放在堆叠上,并在其顶部保持500g重量的支撑。
    11. 允许一夜之间转移,不受干扰。
    12. 第二天拆卸传送组件并将滤膜保持在滤纸上。
    13. 通过紫外线交联(120毫焦耳/厘米2)将RNA固定在膜上1分钟。
    14. 在DEPC处理的水中短暂冲洗膜。
    15. 在室温下将洗涤缓冲液中的膜冲洗5分钟
    16. 在室温下孵育20分钟的1X封闭溶液(见食谱)30分钟 注意:注意不要让膜在此步骤之后的任何时候都干燥。
    17. 在室温下孵育10分钟的抗DIG抗体溶液(1:10,000,1×封闭溶液)中30分钟。抗体来自DIG北方入门套件。
    18. 在室温下平衡100 ml检测缓冲液5 min
    19. 将膜放在开发文件夹上,并应用CDP星(DIG Northern Starter Kit的一部分),并在室温下在黑暗中孵育5分钟。
      注意:清除所有气泡。
    20. 使用凝胶文件系统或使用X光胶片获取化学发光图像。

数据分析

在设置RdRp测定时,应包括适当的对照以解释数据并排除非特异性信号。必须具有阴性对照,例如从反应混合物中省去核苷酸并将RNA酶A包含在反应混合物中。在这两种情况下都不能获得乐队。还应进行使用越来越多的RdRp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RdRp进行测定也可以排除可能的非特异性信号。具有所有对照的样品测定已经在图1中示出。其他因子或化合物对RdRp活性的影响可以通过将它们加入到反应混合物中来评估。在我们的经验中,模板本身不会产生任何错误信号,因为测定依赖于DIG的检测,DIG仅作为DIG-UTP引入RNA。在琼脂糖凝胶中样品迁移期间,从反应中除去游离的DIG-UTP。此外,标记的RNA以约800个碱基对大小迁移,而模板RNA大小为380个碱基对。监测频带的大小也可以使人们对测定的输出有信心。


图1. HEV RNA依赖性RNA聚合酶活性的测定。NTPs:三磷酸核苷酸。

食谱

  1. IP缓存(4°C存储)
    20mM Tris(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)
  2. 5倍试验缓冲液(-20℃保存)
    200mM Tris(pH7.5)
    100mM MgCl 2
    200 mM DTT
    200 mM KCl
    100mM EDTA(pH 8.0)
  3. 2x蛋白酶K缓冲液
    300 mM NaCl
    100mM Tris-Cl(pH7.5)
    1%SDS
  4. 10x MOPS(pH 7.0)
    200 mM MOPS
    50 mM NaAc
    20 mM EDTA(pH 7)
  5. 20x SSC
    3 M NaCl
    300mM柠檬酸钠(pH7)
  6. 马来酸缓冲液(pH 7.5)
    100mM马来酸
    150mM NaCl(pH7.5)
  7. 1x封闭解决方案
    将DIG Northern Starter Kit中的10x阻塞溶液用马来酸缓冲液稀释至1x
    注意:准备新鲜。
  8. 洗涤缓冲液(pH 7.5)
    100mM马来酸
    150 mM NaCl
    0.3%吐温20(pH 7.5)
  9. 检测缓冲液(pH 9.5)
    100mM Tris-Cl
    100mM NaCl(pH9.5)
  10. 含0.1%吐温20(PBST)的磷酸盐缓冲盐水 1x磷酸盐缓冲盐水
    0.1%吐温20

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Behrens等人改编。 (1996)。

参考文献

  1. Behrens,SE,Tomei,L.and De Francesco,R。(1996)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/8598194" target ="_ blank">丙型肝炎病毒的RNA依赖性RNA聚合酶的鉴定和性质 EMBO J 15(1):12-22。
  2. 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。
  3. Emerson,SU,Nguyen,H.,Graff,J.,Stephany,DA,Brockington,A.and Purcell,RH(2004)。< a class ="ke-insertfile"href ="http: ncbi.nlm.nih.gov/pubmed/15078965"target ="_ blank"> E型肝炎病毒(HEV)基因组和表达绿色荧光蛋白的HEV复制子的体外复制。 J Virol 78(9):4838-4846。
  4. 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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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
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., Anang, S., Srivastava, A. and Surjit, M. (2017). RNA-dependent RNA Polymerase Assay for Hepatitis E Virus. Bio-protocol 7(7): e2199. DOI: 10.21769/BioProtoc.2199.
  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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