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Luminescence-based Antiviral Assay for Hepatitis E Virus
针对戊型肝炎病毒的发光抗病毒物质试验   

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Abstract

Hepatitis E virus (HEV) is one of the main causes of acute hepatitis worldwide. Infections are particularly severe in pregnant women and chronic hepatitis E is known to occur in immunocompromised patients. Current therapy (ribavirin or pegylated alpha interferon) has severe side effects and cannot be employed in all patients. In order to evaluate potential new inhibitors of HEV replication, a luminescence-based replicon assay is particularly useful since it offers a rapid read-out and does not pose any biosafety risks (Debing et al., 2014).

Keywords: Hepatitis E virus(戊型肝炎病毒), Antiviral assay(抗病毒活性测定), Subgenomic replicon(亚基因组复制子), Inhibitor(抑制剂)

Materials and Reagents

  1. HEV Kernow-C1 p6/luc plasmid (genotype 3 subgenomic replicon expressing Gaussia luciferase; a kind gift from Suzanne Emerson, NIH) (Shukla et al., 2012)
  2. MluI restriction endonuclease with accompanying 10x buffer D (Promega Corporation, catalog number: R6381 )
  3. pT7-IRES-FFLuc-YFsfRNA plasmid (constructed in-house; IRES, internal ribosome entry site; FFLuc, firefly luciferase; YFsfRNA, yellow fever virus small flaviviral RNA) (Debing et al., 2014)
  4. Primers #1 (5’-CATATGTCGACTAATACGACTCACTATAGGGATCCGCCCCTCTCCC-3’) and #2 (5’-AGTGGTTTTGTGTTTGTCATCC-3’) (custom order) (Integrated DNA Technologies) (dissolved to a final concentration of 10 µM in buffer TE)
  5. Kapa HiFi HotStart ReadyMix master mix (Kapa Biosystems, catalog number: KK2601 )
  6. SeaKem LE agarose for gel electrophoresis (Lonza, catalog number: 50001 )
  7. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
  8. T7 RiboMAX large scale RNA production system (Promega Corporation, catalog number: P1300 )
  9. RNeasy mini kit (QIAGEN, catalog number: 74104 )
  10. ScriptCap m7G capping system (CELLSCRIPTTM, catalog number: C-SCCE0610 )
  11. Huh7 human hepatoma cell line (Japanese Collection of Research Bioresources, catalog number: JCRB0403)
  12. Dulbecco’s modified Eagle’s medium with high glucose (DMEM) (Life Technologies, catalog number: 41965-039 )
  13. Fetal bovine serum (FBS) (not heat-inactivated) (Life Technologies, catalog number: 10270-106 )
  14. Opti-MEM I reduced serum medium (Life Technologies, catalog number: 31985-062 )
  15. Lipofectin transfection reagent (Life Technologies, catalog number: 18292-011 )
  16. Dulbecco’s phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Life Technologies, catalog number: 14190-094 )
  17. Renilla luciferase assay system (Promega Corporation, catalog number: E2810 )
  18. 5x passive lysis buffer (Promega Corporation, catalog number: E1941 ) and demineralized H2O for dilution
  19. Luciferase assay system (Promega Corporation, catalog number: E1501 )
  20. CellTiter 96 AQueous MTS reagent powder [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega Corporation, catalog number: G1111 )
  21. Phenazine methosulphate (PMS) (Sigma-Aldrich, catalog number: P9625 )
  22. Minimum essential medium (MEM) (no glutamine, no phenol red) (Life Technologies, catalog number: 51200-046 )
  23. Tris(hydroxymethyl)aminomethane powder (Trizma base) (Sigma-Aldrich, catalog number: T1503 ) (dissolved to 1 M and pH 8.0 in H2O)
  24. Ethylenediaminetetraacetic acid disodium salt solution (EDTA, 0.5 M) (Sigma-Aldrich, catalog number: E7889 )
  25. MTS/PMS solution (see Recipes)
  26. Buffer TE (see Recipes)

Equipment

  1. Spectrophotometer to determine RNA concentrations (e.g. Thermo Fischer Scientific, NanoDrop, model: ND-1000 )
  2. Falcon transparent 96-well plate (Corning Incorporated, catalog number: 353072 )
  3. CulturPlate white 96-well plate (PerkinElmer, catalog number: 6005680 )
  4. 37 °C and 35 °C 5% CO2 cell culture incubators
  5. Plate shaker
  6. Saphire² microplate reader for both luminescence and absorbance (Tecan Trading AG)

Procedure

  1. 5 µg of the Kernow-C1 p6/luc plasmid is linearized in a 100 µl reaction containing 10 µl of 10x buffer D and 2.5 µl of MluI (10 units/µl) which is incubated at 37 °C for 2 h.
  2. The digested DNA is purified using the Qiaquick gel extraction kit according to the manufacturer’s instructions with final elution in 30 µl of buffer EB.
    Note: To increase the yield, add the recommended amount of isopropanol to the mixture of the reaction and buffer QG. Other methods of DNA purification should be suitable as well.
  3. The T7-IRES-FFLuc-YFsfRNA fragment (2.6 kb) is amplified from the pT7-IRES-FFLuc-YFsfRNA plasmid with primers #1 and #2:
    - 12.5 µl 2x Kapa HiFi HotStart Readymix
    - 0.75 µl of both primers (dissolved at 10 µM)
    - < 0.5 µl of plasmid (1-10 ng of DNA)
    - Ad 25 µl nuclease-free H2O
    Using following temperature scheme:
    - 5 min 95 °C
    - 20” 98 °C    |
    - 15” 65 °C    | x25
    - 3 min 72 °C |
    - 5 min 72 °C
    - 4 °C
  4. The resulting DNA fragment is purified on a 1% agarose gel and extracted with the Qiaquick gel extraction kit according to the manufacturer’s instructions with final elution in 30 µl of buffer EB.
  5. In vitro transcription reactions are prepared for both DNA fragments with the T7 RiboMAX large scale RNA production system:
    - 10 µl of 5x T7 transcription buffer
    - 15 µl of NTPs (ATP, GTP, CTP, UTP; all at 25 mM)
    - 20 µl of DNA template
    - 5 µl of enzyme mix
    Reactions are incubated at 37 °C for 5 h. Afterwards, 5 µl of the supplied RQ1 RNase-free DNase solution is added and incubated at 37 °C for 15 min.
  6. The produced RNA is purified with the RNeasy mini kit according to the manufacturer’s instructions with elution in 60 µl of RNase-free H2O (in 2 elution of 30 µl each). RNA concentration is determined by spectroscopy (absorbance at 260 nm).
  7. Sixty µg of HEV Kernow-C1 p6/luc RNA is capped with the ScriptCap m7G capping system according to the manufacturer’s instructions.
  8. The produced RNA is purified with the RNeasy mini kit according to the manufacturer’s instructions with elution in 60 µl of RNase-free H2O (in 2 elution of 30 µl each). RNA concentration is determined by spectroscopy (absorbance at 260 nm).
  9. Huh7 cells are seeded into a transparent 96-well plate at 104 cells per well in 100 µl of DMEM supplemented with 10% FBS.
  10. Plate is incubated in a 5% CO2 incubator at 37 °C for 24 h.
  11. To transfect one well, 36.5 ng of capped viral RNA and 36.5 ng of FFLuc transfection control RNA are added to 3.65 µl of Opti-MEM. In another tube, 0.365 µl of Lipofectin transfection reagent is added to 3.65 µl Opti-MEM. Both tubes are incubated at room temperature for 30 min. Then, the contents of both tubes are combined and incubated for another 10 min at room temperature. An additional 29.2 µl of Opti-MEM is added and the resulting solution is mixed well.
    Note: Multiply the indicated amounts and volumes by the number of wells to transfect and prepare an additional 10% extra. For cell control (CC) wells without replicon, viral RNA is omitted.
  12. The culture medium is removed from cells seeded earlier and each well is washed with 100 µl of DMEM.
    Note: Huh7 cells may easily detach from the culture plate, so medium is removed carefully using a pipette instead of a vacuum system.
  13. To each well, 36.5 µl of transfection mixture is added.
  14. Plate is incubated in a 5% CO2 incubator at 37 °C for 5 h.
  15. Transfection medium is removed from each well and cell layers are washed once with 100 µl of PBS.
  16. One hundred microliters of DMEM supplemented with 10% FBS is added to each well. At this step, the compound to be tested is added and an appropriate dilution series is made.
    Note: No compound is added to virus control (VC) and CC wells.
  17. Plate is incubated in a 5% CO2 incubator at 35 °C for 72 h.
    Note: Incubation can also be performed at 37 °C if no incubator would be available at 35 °C.
  18. From each well, 20 µl is transferred to a white CulturPlate.
  19. Renilla luciferase substrate is diluted 1: 100 in assay buffer and 50 µl is added to each well of the CulturPlate containing the transferred culture medium.
  20. The plate is shaken vigorously on a plate shaker for a few seconds and luminescence read-out is performed (integration time: 100 ms).
    Note: The time between substrate addition and read-out has to be kept to a minimum, since background signal will increase over time during the first few minutes. Later on, the overall signal will decline.
  21. The remaining medium is removed from each well of the transfected 96-well plate.
  22. Passive lysis buffer is diluted 1: 5 in demineralized H2O and 20 µl is added to each well.
  23. Plate is shaken at room temperature for 10 min at 500 rpm for proper cell lysis.
  24. One hundred microliters of reconstituted Firefly luciferase substrate solution is added to the wells of a new white CulturPlate.
  25. All of the cell lysate solution is transferred into the luciferase substrate solution in the CulturPlate.
  26. The plate is shaken vigorously on a plate shaker for a few seconds and luminescence read-out is performed as described above.
  27. In order to assess compound toxicity, the following assay can be performed:
    1. Huh7 cells are seeded as described and incubated in a 5% CO2 incubator at 37 °C for 24 h.
    2. Culture medium is replaced by a compound dilution series in fresh DMEM supplemented with 10% FBS (100 µl per well). Plate is incubated in a 5% CO2 incubator at 35 °C for 72 h.
    3. Medium is removed and replaced with 100 µl of a 1: 20 dilution of MTS/PMS solution in colorless MEM per well. Plate is incubated in a 5% CO2 incubator at 37 °C until a brownish color develops (usually this requires incubation for 1-2 h).
    4. Absorbance read-out is performed by determining the optical density (OD) at 498 nm (OD of CC should be above 0.6 but below 1 to stay in the linear range).

Representative data

  1. A possible outline for a 96-well plate may be the following:



  2. A typical read-out for the luminescence-based assay would be the following:


    p6/luc-transfected wells
    CC wells
    Relative luminescence units (RLU)
    85264
    759

    The variability in Gaussia luminescence read-out between different replicates of the same experiment might be rather large. This is resolved by normalizing data to those obtained for untreated VC wells.

Notes

  1. The T7-IRES-FFLuc-YFsfRNA is used as a transfection control to normalize for transfection efficacy (see calculations below). Other FFluc-expressing RNA fragments can be used as well. The transfection control can be omitted, although this may impact the assay’s robustness.
  2. Following formulas are used to calculate the antiviral activity (%). First, Gaussia luciferase signals are normalized:
    LucNormcompound = (GLucreplicon+compound – GlucCC)/FFLucreplicon+compound
    LucNormVC = (GlucVC – GlucCC)/FFLucVC
    Next, antiviral activity is calculated:
    % Antiviral activity = 100 - (LucNormcompound/LucNormVC x 100)
    For example, if the following luminescence data would have been obtained:


    Compound
    VC
    CC
    Gluc
    18524
    85264
    759
    FFLuc
    10362
    9254
    8906

    Antiviral activity is calculated as follows:
    LucNormcompound = (18524 - 759)/10362 = 1.71
    LucNormVC = (85264 - 759)/9254 = 9.13
    % Antiviral activity = 100 - (1.71/9.13 x 100) = 81.3%
  3. To calculate cell viability, the following formula can be employed:
    % Viability = ODcompound/ODCC x 100
  4. The same protocol can be used with the Sar55/S17/luc construct, a genotype 1 subgenomic HEV replicon (Nguyen et al., 2014).

Recipes

  1. MTS/PMS solution
    1. Two grams of MTS powder is dissolved in 1 L of PBS and stirred for 15 min.
    2. Next, 46 mg of PMS powder is added (pH should be between 6-6.5).
    3. Solution is filtered, aliquoted and stored at -20 °C.
    Note: Since MTS is light-sensitive, the powder and solution should be protected from light.
  2. Buffer TE
    To 25 ml of nuclease-free H2O, add
    250 µl of a 1 M Tris solution (pH 8.0) (final concentration: 10 mM)
    50 µl of a 0.5 M EDTA solution (final concentration: 1 mM)

Acknowledgments

This protocol was adapted from Debing et al. (2014) and is partially based on earlier work by Shukla et al. (2012). Yannick Debing is a fellow of the Research Foundation-Flanders (FWO). This work was supported by KU Leuven Geconcerteerde Onderzoeksacties (GOA/10/014) and by EU FP7 project SILVER (260644).

References

  1. Debing, Y., Emerson, S. U., Wang, Y., Pan, Q., Balzarini, J., Dallmeier, K., Neyts, J. (2014). Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon. Antimicrob Agents Chemother 58(1): 267-273.
  2. Nguyen, H.T., Shukla, P., Torian, U., Faulk, K., Emerson, S.U. (2014) Hepatitis E virus genotype 1 infection of swine kidney cells in vitro is inhibited at multiple levels. J Virol 88(2): 868-877.
  3. Shukla, P., Nguyen, H.T., Faulk, K., Mather, K., Torian, U., Engle, R.E., Emerson, S.U. (2012) Adaptation of a genotype 3 hepatitis E virus to efficient growth in cell culture depends on an inserted human gene segment acquired by recombination. J Virol 86(10): 5697-5707.

简介

戊型肝炎病毒(HEV)是全世界急性肝炎的主要原因之一。 感染在孕妇中特别严重,已知慢性戊型肝炎发生在免疫受损的患者中。 目前的治疗(利巴韦林或聚乙二醇化α干扰素)具有严重的副作用,不能用于所有患者。 为了评价潜在的新的HEV复制抑制剂,基于发光的复制子测定法是特别有用的,因为其提供了快速读出,并且不会引起任何生物安全风险(Debing等人,2014) 。

关键字:戊型肝炎病毒, 抗病毒活性测定, 亚基因组复制子, 抑制剂

材料和试剂

  1. HEV Kernow-C1 p6/luc质粒(表达Gaussia荧光素酶的基因型3亚基因组复制子;来自Suzanne Emerson,NIH的赠品)(Shukla等人,2012)
  2. MluI限制性内切酶与伴随的10x缓冲液D(Promega Corporation,目录号:R6381)
  3. pT7-IRES-FFLuc-YFsfRNA质粒(内部构建; IRES,内部核糖体进入位点; FFLuc,萤火虫荧光素酶; YFsfRNA,黄热病毒小黄病毒RNA)(Debing等人,
  4. 引物#1(5'-CATATGTCGACTAATACGACTCACTATAGGGATCCGCCCCTCTCCC-3')和#2(5'-AGTGGTTTTGTGTTTGTATG-3')(定制序列)(Integrated DNA Technologies)(在缓冲液TE中溶解至终浓度为10μM)
  5. Kapa HiFi HotStart ReadyMix主混合物(Kapa Biosystems,目录号:KK2601)
  6. 用于凝胶电泳的SeaKem LE琼脂糖(Lonza,目录号:50001)
  7. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  8. T7 RiboMAX大规模RNA生产系统(Promega Corporation,目录号:P1300)
  9. RNeasy迷你试剂盒(QIAGEN,目录号:74104)
  10. ScriptCap m7G加盖系统(CELLSCRIPT TM ,目录号:C-SCCE0610)
  11. Huh7人肝癌细胞系(Japanese Collection of Research Bioresources,目录号:JCRB0403)
  12. 具有高葡萄糖的Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,目录号:41965-039)
  13. 胎牛血清(FBS)(非热灭活)(Life Technologies,目录号:10270-106)
  14. Opti-MEM I还原血清培养基(Life Technologies,目录号:31985-062)
  15. Lipofectin转染试剂(Life Technologies,目录号:18292-011)
  16. 不含Ca 2+和Mg 2+的Dulbecco's磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14190-094)
  17. 萤光素酶测定系统(Promega公司,目录号:E2810)
  18. 5x被动裂解缓冲液(Promega Corporation,目录号:E1941)和用于稀释的去矿质H 2 O 2。
  19. 荧光素酶测定系统(Promega Corporation,目录号:E1501)
  20. CellTiter 96 AQueous MTS试剂粉末[3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑](Promega Corporation,目录号:G1111)
  21. 吩嗪硫酸甲酯(PMS)(Sigma-Aldrich,目录号:P9625)
  22. 最低必需培养基(MEM)(无谷氨酰胺,无酚红)(Life Technologies,目录号:51200-046)
  23. 三(羟甲基)氨基甲烷粉末(Trizma碱)(Sigma-Aldrich,目录号:T1503)(溶解至1M,pH8.0,在H 2 O中)
  24. 乙二胺四乙酸二钠盐溶液(EDTA,0.5M)(Sigma-Aldrich,目录号:E7889)
  25. MTS/PMS解决方案(参见配方)
  26. 缓冲TE(参见配方)

设备

  1. 用于确定RNA浓度的分光光度计(例如 Thermo Fischer Scientific,NanoDrop,型号:ND-1000)
  2. Falcon透明96孔板(Corning Incorporated,目录号:353072)
  3. 培养平板白色96孔板(PerkinElmer,目录号:6005680)
  4. 37℃和35℃5%CO 2细胞培养孵化器
  5. 板振动器
  6. 用于发光和吸光度的Saphire²酶标仪(Tecan Trading AG)

程序

  1. 在含有10μl10×缓冲液D和2.5μlMluI(10单位/μl)的100μl反应物中将5μgKernow-C1 p6/luc质粒线性化,其在37℃温育2小时。 >
  2. 使用Qiaquick凝胶提取试剂盒根据制造商的说明纯化消化的DNA,在30μl缓冲液EB中最终洗脱。
    注意:为了提高产量,向反应和缓冲液QG的混合物中加入推荐量的异丙醇。其他DNA纯化方法也应该适用。
  3. 使用引物#1和#2从pT7-IRES-FFLuc-YFsfRNA质粒扩增T7-IRES-FFLuc-YFsfRNA片段(2.6kb):
    - 12.5μl2x Kapa HiFi HotStart Readymix
    - 0.75μl两种引物(溶解于10μM)
    - - 0.5μl质粒(1-10ng DNA)
    - 加入25μl不含核酸酶的H 2 O 2/b 使用以下温度方案:
    - 5分钟95℃
    - 20"98°C    |
    - 15"65°C    | x25
    - 3分钟72°C |
    - 5分钟72℃
    - 4°C
  4. 所得DNA片段在1%琼脂糖凝胶上纯化,并根据制造商的说明书用Qiaquick凝胶提取试剂盒提取,最后在30μl缓冲液EB中洗脱。
  5. 使用T7 RiboMAX大规模RNA生产系统为两个DNA片段制备体外转录反应:
    - 10μl5x T7转录缓冲液
    - 15μlNTP(ATP,GTP,CTP,UTP;全部为25mM) - 20μlDNA模板
    - 5μl酶混合物
    将反应在37℃孵育5小时。之后,加入5μl提供的RQ1 RNase-free DNase溶液,并在37℃下孵育15分钟。
  6. 根据制造商的说明书,使用RNeasy微型试剂盒纯化所产生的RNA,在60μl无RNase的H 2 O(在2次洗脱中各30μl)中洗脱。通过光谱法(260nm处的吸光度)测定RNA浓度
  7. 根据制造商的说明书,用ScriptCap m7G帽系统覆盖60μgHEV Kernow-C1 p6/luc RNA。
  8. 根据制造商的说明书,使用RNeasy微型试剂盒纯化所产生的RNA,在60μl无RNase的H 2 O(在2次洗脱中各30μl)中洗脱。通过光谱法(260nm处的吸光度)测定RNA浓度
  9. 将Huh7细胞以每孔10 10个细胞接种在100μl补充有10%FBS的DMEM中的透明96孔板中。
  10. 将板在5%CO 2培养箱中在37℃孵育24小时
  11. 为了转染一个孔,将36.5ng加帽的病毒RNA和36.5ng的FFLuc转染对照RNA加入3.65μlOpti-MEM中。在另一个管中,将0.365μl的Lipofectin转染试剂加入到3.65μlOpti-MEM中。将两管在室温下温育30分钟。然后,将两个管的内容物合并,并在室温下再温育10分钟。再加入29.2μlOpti-MEM,将所得溶液充分混合 注意:将指示的量和体积乘以孔的数量,以转染并额外补充10%。对于没有复制子的细胞对照(CC)孔,省略病毒RNA。
  12. 从早期接种的细胞中除去培养基,每孔用100μlDMEM洗涤 注意:Huh7细胞可能容易从培养板上脱落,因此使用移液管而不是真空系统小心地除去培养基。
  13. 向每个孔中加入36.5μl转染混合物
  14. 将板在5%CO 2培养箱中在37℃下孵育5小时
  15. 从每个孔中除去转染培养基,并用100μlPBS洗涤细胞层一次
  16. 向每个孔中加入100微升补充有10%FBS的DMEM。 在该步骤中,加入待测试的化合物,并进行适当的稀释系列 注意:没有化合物添加到病毒控制(VC)和CC井。
  17. 将板在5%CO 2培养箱中在35℃温育72小时 注意:如果在35°C没有孵化器,也可以在37°C孵育。
  18. 从每个孔中,将20μl转移至白色培养板
  19. 将海肾荧光素酶底物在测定缓冲液中以1:100稀释,并将50μl加入含有转移的培养基的CulturPlate的每个孔中。
  20. 将板在板振荡器上剧烈摇动几秒钟并进行发光读出(积分时间:100ms)。
    注意:基底添加和读出之间的时间必须保持最小,因为背景信号将在最初几分钟内随时间增加。后来,整体信号会下降。
  21. 从转染的96孔板的每个孔中除去剩余的培养基
  22. 将被动裂解缓冲液在去离子H 2 O中以1:5稀释,并向每个孔中加入20μl。
  23. 将板在室温下在500rpm下摇动10分钟以进行适当的细胞裂解
  24. 将100微升重组的萤火虫荧光素酶底物溶液加入新的白色CulturPlate的孔中。
  25. 将所有细胞裂解物溶液转移到CulturPlate中的荧光素酶底物溶液中
  26. 将板在板振荡器上剧烈摇动几秒钟,并如上所述进行发光读出。
  27. 为了评估化合物毒性,可以进行以下测定:
    1. 如所述接种Huh7细胞,并在5%CO 2培养箱中在37℃温育24小时。
    2. 培养基用补充有10%FBS(每孔100μl)的新鲜DMEM中的化合物稀释系列代替。将板在5%CO 2培养箱中在35℃温育72小时
    3. 除去培养基并用100μl1:20稀释的MTS/PMS溶液(无色MEM /孔)代替。将板在5%CO 2培养箱中在37℃温育,直到出现褐色(通常这需要孵育1-2小时)。
    4. 通过确定498nm处的光密度(OD)进行吸光度读数(CC的OD应该高于0.6但低于1以保持在线性范围内)。

代表数据

  1. 96孔板的可能轮廓可能如下:



  2. 基于发光的测定的典型读数如下:


    p6/luc转染孔
    CC井
    相对发光单位(RLU)
    85264
    759

    在相同实验的不同重复之间的Gaussia 发光读数的变异性可能相当大。 这可以通过将数据归一化为未处理的VC井获得的数据来解决

笔记

  1. T7-IRES-FFLuc-YFsfRNA用作转染对照以使转染效率正常化(参见下面的计算)。 也可以使用其它表达FFluc的RNA片段。 可以省略转染控制,尽管这可能影响测定的稳健性
  2. 以下公式用于计算抗病毒活性(%)。 首先,将 Gaussia 荧光素酶信号标准化:
    LucNorm 化合物 =(GLuc复制子+化合物-Gluc CC)/FFLuc复制子+化合物
    LucNorm VC =(Gluc VC - Gluc CC )/FFLuc 接下来,计算抗病毒活性:
    %抗病毒活性= 100-(LucNorm化合物/LucNorm VC×100)
    例如,如果将获得以下发光数据:


    化合物
    VC
    CC
    Gluc
    18524
    85264
    759
    FFLuc
    10362
    9254
    8906,

    抗病毒活性计算如下:
    LucNorm compound =(18524 - 759)/10362 = 1.71
    LucNorm VC =(85264 - 759)/9254 = 9.13
    %抗病毒活性= 100 - (1.71/9.13×100)= 81.3%
  3. 为了计算细胞存活率,可以采用以下公式:
    %活力= OD化合物/OD CC×100
  4. 相同的方案可以用于Sar55/S17/luc构建体,基因型1亚基因组HEV复制子(Nguyen等人,2014)。

食谱

  1. MTS/PMS溶液
    1. 将2克MTS粉末溶解在1升PBS中并搅拌15分钟
    2. 接下来,加入46mg PMS粉末(pH应在6-6.5之间)
    3. 将溶液过滤,分装并储存在-20℃
    注意:由于MTS是光敏的,所以粉末和溶液应该避光。
  2. 缓冲区TE
    向25ml无核酸酶的H 2 O 2中加入
    250μl1M Tris溶液(pH8.0)(终浓度:10mM) 50μl0.5M EDTA溶液(终浓度:1mM)

致谢

该协议改编自Debing等人(2014),并且部分基于Shukla等人早期的工作(2012)。 Yannick Debing是研究基金会佛兰德斯(FWO)的研究员。这项工作由KU Leuven Geconcerteerde Onderzoeksacties(GOA/10/014)和欧盟FP7项目SILVER(260644)支持。

参考文献

  1. Debing,Y.,Emerson,S.U.,Wang,Y.,Pan,Q.,Balzarini,J.,Dallmeier,K.,Neyts,J。(2014)。 利巴韦林通过消耗细胞GTP库来抑制体外的艾滋病病毒复制[/em]并且与α干扰素具有中等协同作用。 Antimicrob Agents Chemother 58(1):267-273。
  2. Nguyen,H.T.,Shukla,P.,Torian,U.,Faulk,K.,Emerson,S.U。 (2014)体外对猪肾细胞的E型肝炎病毒基因型1感染在多个水平受到抑制。 J Virol 88(2):868-877。
  3. Shukla,P.,Nguyen,H.T.,Faulk,K.,Mather,K.,Torian,U.,Engle,R.E.,Emerson, (2012)基因型3戊型肝炎病毒适应 细胞培养物中的有效生长取决于通过重组获得的插入的人基因区段。 J Virol 86(10):5697-5707。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Debing, Y., Dallmeier, K. and Neyts, J. (2014). Luminescence-based Antiviral Assay for Hepatitis E Virus. Bio-protocol 4(15): e1196. DOI: 10.21769/BioProtoc.1196.
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