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Transient Transfection-based Fusion Assay for Viral Proteins
基于瞬时转染的病毒蛋白融合分析法   

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Abstract

Membrane fusion is vital for entry of enveloped viruses into host cells as well as for direct viral cell-to-cell spread. To understand the fusion mechanism in more detail, we use an infection free system whereby fusion can be induced by a minimal set of the alphaherpesvirus pseudorabies virus (PrV) glycoproteins gB, gH and gL. Here, we describe an optimized protocol of a transient transfection based fusion assay to quantify cell-cell fusion induced by the PrV glycoproteins.

Keywords: Herpesvirus(疱疹病毒), Pseudorabies virus(伪狂犬病病毒), Membrane fusion(膜融合), Virus entry(病毒侵入), Glycoproteins gB(糖蛋白gB), gH/gL(gH/gL), Transfection(转染)

Background

Membrane fusion is essential for entry and spread of enveloped viruses. Many enveloped viruses require only one or two viral proteins to mediate attachment to host cells and membrane fusion, and the molecular mechanisms are well understood (Harrison, 2015). In contrast, herpesviruses use a more complex mechanism requiring a receptor-binding protein and the core fusion machinery composed of gB and the heterodimeric gH/gL complex for infectious entry. The mechanism leading to fusion of herpesvirus envelopes with cellular membranes is only incompletely understood. Detailed knowledge of the molecular basis of herpesvirus entry and spread is important for efficient countermeasures against a variety of diseases. A better understanding is aided by studying the cell fusion activity of cells transiently expressing the relevant proteins. Different model systems, whereby fusion is induced with a minimal set of the core fusion machinery represented by glycoproteins gB and gH/gL and receptor-binding gD, in the absence of infection, have been developed, for example, for herpes simplex viruses type 1 and 2 (HSV-1 and 2 [Turner et al., 1998; Muggeridge, 2000; McShane and Longnecker, 2005]). Unlike HSV-1 and 2, PrV does not require signaling of gD for membrane fusion during direct cell-to-cell spread, reducing the number of relevant proteins to three (Schmidt et al., 1997). These systems are also used to quantify membrane fusion. However, the evaluation or quantitation of fusion activity, which is often based on counting the number of nuclei of a formed syncytium, is very time consuming. Our incentive to develop the present protocol was to improve the current protocol to facilitate and accelerate evaluation, and make results more robust and comparable by combining important factors like size and number of formed syncytia. Here, we describe an optimized protocol for an in vitro transient transfection based cell-cell fusion assay to quantify membrane fusion induced by the PrV glycoproteins gB and gH/gL (Schröter et al., 2015). However, we know that this assay also is functional with other fusion-active glycoproteins, not just those of pseudorabies virus.

Materials and Reagents

  1. 1.5 ml tubes (e.g., Fisher Scientific, catalog number: S348903 )
  2. 24-well cell culture plate (e.g., Corning, Costar®, catalog number: 3527 )
  3. Pipette tips 10 µl (TipOne) (STARLAB INTERNATIONAL, catalog number: S1110-3000 )
  4. Pipette tips 1 ml (Greiner Bio One International, catalog number: 740290 )
  5. Pipette tips 100 µl (Greiner Bio One International, catalog number: 739290 )
  6. pcDNA3 (Thermo Fisher Scientific, Invitrogen)
    Note: pcDNA3 from Invitrogen is no longer available. Alternatively, pcDNA3.1(+) (Thermo Fisher Scientific, catalog number: V79020 ) can be used.
  7. Rabbit kidney (RK13) cells (Collection of Cell Lines in Veterinary Medicine-RIE 109)
  8. MEM Eagle (Hank’s salts and L-glutamine) (Sigma-Aldrich, catalog number: M4642 )
  9. MEM (Earle’s salts) (Thermo Fisher Scientific, catalog number: 61100061 )
  10. Sodium bicarbonate (NaHCO3) (Carl Roth, catalog number: 6885.1 )
  11. NEA (nonessential amino acids) (Biochrom, catalog number: K 0293 )
  12. Na-pyruvate (EMD Millipore, catalog number: 106619 )
  13. Tris-HCl (pH 8.5)
  14. Fetal bovine serum (FBS) (Biowest, catalog number: S181G )
  15. Lipofectamine® 2000 reagent (Thermo Fisher Scientific, catalog number: 11668027 )
  16. Opti-MEM® reduced serum medium (Thermo Fisher Scientific, catalog number: 31985062 )
  17. Sodium chloride (NaCl) (Carl Roth, catalog number: 9265.1 )
  18. Potassium chloride (KCl) (Carl Roth, catalog number: 5346.1 )
  19. Dextrose (Sigma-Aldrich, catalog number: D9434 )
  20. Trypsin (1:250) powder (Thermo Fisher Scientific, catalog number: 27250018 )
  21. Ethylenediaminetetraacetic acid (EDTA) (SERVA Electrophoresis, catalog number: 11280.01 )
  22. Paraformaldehyde (Carl Roth, catalog number: 0335.1 )
  23. 10% growth media (see Recipes)
  24. 3% paraformaldehyde (see Recipes)
  25. 10 mM Tris HCl (pH 8.5) (see Recipes)
  26. Alsever’s-Trypsin Versen (ATV-) solution (see Recipes)

Note: Solutions and media #6, 11, 14, 15, 16, 21, 22, 23, 24 and 26 should be kept at 4 °C.

Equipment

  1. T75 flask (e.g., Corning, catalog number: 430725U )
  2. Incubator (e.g., Panasonic, Sanyo, model: MCO-19AIC or Fisher Scientific, catalog number: 12826756 )
  3. Fluorescence microscope (e.g., Nikon Instruments, model: Eclipse Ti-S)
  4. Super high pressure mercury lamp power supply (e.g., Nikon Instruments, model: C-SCH1 )
  5. Light source (e.g., Nikon Instruments, model: LH-M100C-1 )
  6. Vortexer (e.g., Fisher Scientific, catalog number: S96461A )
  7. Laminar flow hood (Thermo Fisher Scientific, Thermo ScientificTM, model: Safe 2020 Class II , catalog number: 51026637)
  8. Centrifuges for 1.5 ml tubes (e.g., Eppendorf, model: 5415 D )
  9. Pipette controller (e.g., pipetboy acu 2, VWR, catalog number: 37001-856 )
  10. Nanophotometer (e.g., VWR, IMPLEN, model: P330, catalog number: CA11027-294 )

Software

  1. Computer running software NIS-Elements V 4.00.01 (Nikon, Düsseldorf, Germany)

Procedure

  1. Preparation of plasmids
    1. Dilute the plasmids expressing the putative fusion proteins to 200 ng/µl with Tris-HCl pH 8.5 (see Recipes). Include a plasmid expressing an autofluorescent protein (e.g., GFP or mCherry) as a marker to facilitate evaluation of the assay. Here, a concentration of 100 ng/µl is sufficient.
      Note: The expression plasmids we use for our experiments are based on pcDNA3, a mammalian expression vector with constitutive transgene expression under control of the human cytomegalovirus immediate-early 1 promoter/enhancer complex and a neomycin-resistance pEGFP-N1 as marker to facilitate evaluation of the assay (Schröter et al., 2015).

  2. Preparation of cells
    1. Grow rabbit kidney (RK13) cells in Eagle’s minimum essential medium supplemented with 10% FBS. Trypsinize cells with ATV (Recipe 3) and seed ~1.8 x 105 cells per well onto 24-well cell culture dishes.
    2. On the following day, use cells for transfection. Cells should have a confluency of 80-90%.
      Note: Cell confluency for optimal transfection efficiency varies depending on the cell type. For RK13 and Vero cells 80-90% confluency is suggested.

  3. Transfection
    1. Prepare DNA-mixture and Lipofectamine-mixture in separate tubes.
      1. DNA-mixture:
        Use 200 ng of each of the expression plasmids and dilute the DNA-mixture in 50 µl Opti-MEM.
      2. Lipofectamine-mixture:
        Use 1 µl Lipofectamine 2000TM in 50 µl Opti-MEM per well. Add Lipofectamine drop-wise to 50 µl Opti-MEM and mix gently. Incubate Lipofectamine-mixture for 5 min at room temperature.
        Note: The optimal amount of Lipofectamine varies depending on the cell type. For RK13 and Vero cells 1 µl per 24-well is suggested.
    2. Add Lipofectamine-mixture drop-wise into DNA-mixture, gently tap the solution and incubate for 20 min at room temperature (RT) (Video 1).
      Note: It is important to mix gently but thoroughly to allow formation of the DNA-lipid complex.

      Video 1. Tutorial: How to add Lipofectamine-mixture drop-wise to DNA-mixture

    3. Remove media from the cells and add the transfection-solution drop-wise directly to the cell-monolayer (Video 2).
      Note: To allow equal distribution of the transfection-solution add it circularly drop-by-drop onto the monolayer (Video 2).

      Video 2. Tutorial: How to add transfection solution to the cell-monolayer

    4. Add an additional 200 µl Opti-MEM to the cells to prevent drying of cells.
    5. Incubate cells at 37 °C and 5% CO2 for 3 h.
    6. After 3 h, remove the transfection-solution from the cells and add fresh MEM supplemented with 2% FBS to the cells and incubate them 18-24 h at 37 °C and 5% CO2. For an overview of the whole procedure see Figure 1.
      Note: To achieve comparable results, use exactly the same incubation time in each experiment before fixing the cells.


      Figure 1. Schematic presentation of the transfection procedure

  4. Fixation
    1. Prepare 3% PFA (see Recipes).
    2. Remove media and wash cell monolayer for 2 min with ~1 ml PBS per well using a rocker at RT.
    3. Remove PBS and fix cells with 200 µl 3% PFA per well. Fix cells for 20 min at RT.
    4. Wash cells 2 x with 1 ml PBS per well.
    5. Add 1 ml PBS per well and store cells at 4 °C.

  5. Determination of fusion activity
    Note: To quantify fusion activity the number and the area of green-fluorescing syncytia with three or more nuclei is measured using a Nikon Eclipse Ti-S fluorescence microscope and Nikon NIS-Elements imaging software.
    1. Use fixed cells for determination of fusion activity by fluorescence microscopy.
    2. Open NIS-Elements imaging software (Nikon) and select corresponding type of fluorescence and objective (e.g., FITC, 10x) (see Figure 2 red boxes).
    3. Select Measure  Manual Measurement  Area (Figure 2A, Video 3).
      Note: NIS-Elements supports units including: pixels, nanometers, micrometers, millimeters, centimeters, decimeters, meters, inches, and mils. For our measurements we use micrometers.


      Figure 2. Example view of NIS Elements software. A. Selecting manual measurement; B. Exporting data.
      Note: First, measure the area of syncytia on the right-hand corner of the screen, since each syncytium will be marked with a white icon on its right site, which can hinder the view over syncytia to the right of it (see Figure 2B).

    4. Measure all apparent syncytia in 10 fields of view (~5.5 mm2 each), each time starting in the top right-hand corner of the corresponding image. The data, including number of each syncytium as well as mean syncytia area will appear in a table on the lower right-hand corner (see Figure 2B green box, Video 3).

      Video 3. Tutorial: How to measure syncytia with NIS Elements software

    5. Export the data directly to an Excel file or a text file by clicking the export options button (see Figure 3 red box). (Select whether to export the data to an Excel file or a text file.) You can modify the name and path of the file via the browse button.
    6. Repeat the entire experiment at least three times on three different days to calculate the relative fusion activity and corresponding standard deviations (SD).

Data analysis

  1. To calculate the total fusion activity for each sample, corresponding to a given well, multiply the number of syncytia with the mean syncytia area of the 10 fields of view.
    E.g., you counted 107 syncytia and the mean syncytia area was 7,048 µm2, the fusion activity would be 107 x 7,048.6 = 754,200.2.
  2. Calculate the standard deviation (SD) of the total fusion activity of each sample by comparing the fusion activities estimated for each of the three independent experiments.
  3. The relative fusion activity of the different samples is determined by comparing their total fusion activity to the positive control (e.g., wild-type gB, gH and gL), which should be set as 100%.
    E.g., if the total fusion activity of the positive control is 754,200.2 and the fusion activity of the test sample was 431,708.6, the relative fusion activity of the test sample would be (100/754,200.2) x 431,708.6 = 57% (Figure 3).
    Note: Use Excel/Prism or comparable tools to facilitate calculations and create graphs.
  4. Representative data


    Figure 3. Fusion activity. To test for fusion activity RK13 cells were cotransfected with expression plasmids encoding wild-type gB or mutated gB, and gH, gL and gD as well as EGFP to facilitate evaluation of the assay by fluorescence microscopy. Assays conducted with a plasmid encoding wild-type gB in combination with wild-type gH, gL and gD were set as 100%. Assays with the empty expression vector pcDNA3 served as negative control. One day after transfection, the areas of cells containing three or more nuclei were measured and multiplied by the number of syncytia to determine the fusion activity. Corresponding standard deviations for three independent assays were calculated.

Recipes

  1. 10% growth media (1 L), pH 7.2
    The following reagents should be dissolved in double distilled water to a final volume of 1 L:
    5.32 g MEM Eagle (Hank’s salt)
    4.76 g MEM (Earle’s salt)
    1.25 g NaHCO3
    10 ml NEA
    120 mg Na-pyruvate
  2. 10 mM Tris-HCl, pH 8.5
    Prepare 2 M stock solution (500 ml):
    121.14 g Tris HCl
    Adjust pH to 8.5 with ~50 ml fuming HCl
    Dilute stock solution 1:20 with ddH2O
  3. Alsever’s-Trypsin Versen (ATV-)solution, pH 7.2
    8.5 g NaCl
    0.4 g KCl
    1.0 g dextrose
    0.58 g NaHCO3
    0.5 g trypsin 1:250
    0.2 g EDTA
    Ad 1 L ddH2O
  4. 3% PFA (100 ml)
    3 g paraformaldehyde
    100 ml PBS
    Let PFA dissolve at 65 °C

Acknowledgments

This protocol was adapted and modified from a previous study (Schroter et al., 2015). This work was supported by grants from DFG (ME 854/11).

References

  1. Harrison, S. C. (2015). Viral membrane fusion. Virology 479-480: 498-507.
  2. Turner, A., Bruun, B., Minson, T. and Browne, H. (1998). Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J Virol 72(1): 873-875.
  3. Muggeridge, M. I. (2000). Characterization of cell-cell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells. J Gen Virol 81(Pt 8): 2017-2027.
  4. McShane, M. P. and Longnecker, R. (2005). Analysis of fusion using a virus-free cell fusion assay. Methods Mol Biol 292: 187-196.
  5. Schmidt, J., Klupp, B. G., Karger, A. and Mettenleiter, T. C. (1997). Adaptability in herpesviruses: glycoprotein D-independent infectivity of pseudorabies virus. J Virol 71(1): 17-24.
  6. Schröter, C., Vallbracht, M., Altenschmidt, J., Kargoll, S., Fuchs, W., Klupp, B. G. and Mettenleiter, T. C. (2015). Mutations in pseudorabies virus glycoproteins gB, gD, and gH functionally compensate for the absence of gL. J Virol 90(5): 2264-2272.

简介

膜融合对于将包膜病毒进入宿主细胞以及直接病毒细胞到细胞传播至关重要。为了更好地了解融合机制,我们使用无感染系统,可以通过最小的α疱疹病毒伪狂犬病病毒(PrV)糖蛋白gB,gH和gL诱导融合。在这里,我们描述了基于瞬时转染的融合测定的优化方案,以定量由PrV糖蛋白诱导的细胞 - 细胞融合。

背景 膜融合对于包膜病毒的进入和扩散至关重要。许多包膜病毒只需要一种或两种病毒蛋白来介导宿主细胞的附着和膜融合,分子机制也被很好地理解(Harrison,2015)。相比之下,疱疹病毒使用需要受体结合蛋白的更复杂的机制和由gB和异二聚体gH / gL复合物组成的核心融合机构用于感染性进入。导致疱疹病毒包膜与细胞膜融合的机制尚不完全清楚。疱疹病毒进入和传播的分子基础的详细知识对于针对各种疾病的有效对策是重要的。通过研究瞬时表达相关蛋白质的细胞的细胞融合活性来帮助更好的理解。已经开发了不同的模型系统,其中在不存在感染的情况下,开发了由糖蛋白gB和gH / gL和受体结合gD表示的最小的核心融合机制的融合,例如,对于1型单纯疱疹病毒和2(HSV-1和2 [Turner等人,1998; Muggeridge,2000; McShane和Longnecker,2005])。与HSV-1和2不同,PrV在直接细胞间传播期间不需要gD信号传导用于膜融合,将相关蛋白的数量减少到3个(Schmidt等人,1997)。这些系统也用于定量膜融合。然而,融合活性的评估或定量通常是基于计数形成的合胞体的细胞核数量,这是非常耗时的。我们制定本议定书的动机是改进目前的方案,以促进和加速评估,并通过组合形成合成细胞的数量和数量等重要因素,使结果更加强大和可比。在这里,我们描述了用于定量由PrV糖蛋白gB和gH / gL诱导的膜融合的基于瞬时转染的细胞 - 细胞融合测定的体外优化方案(Schröter等人>。,2015)。然而,我们知道,该测定法也与其他融合活性糖蛋白功能不仅仅是伪狂犬病毒的功能。

关键字:疱疹病毒, 伪狂犬病病毒, 膜融合, 病毒侵入, 糖蛋白gB, gH/gL, 转染

材料和试剂

  1. (例如,Fisher Scientific,目录号:S348903)
  2. 24孔细胞培养板(例如,Corning,Costar ,目录号:3527)
  3. 移液管吸头10μl(TipOne)(STARLAB INTERNATIONAL,目录号:S1110-3000)
  4. 移液器提示1 ml(Greiner Bio One International,目录号:740290)
  5. 移液器提示100μl(Greiner Bio One International,目录号:739290)
  6. pcDNA3 (Thermo Fisher Scientific,Invitrogen)
    注意:来自Invitrogen的pcDNA3不再可用。或者,可以使用pcDNA3.1 (+)(Thermo Fisher Scientific,目录号:V79020)。
  7. 兔肾(RK13)细胞(兽医学报收集的RIE 109)
  8. MEM Eagle(汉克盐和L-谷氨酰胺)(Sigma-Aldrich,目录号:M4642)
  9. MEM(Earle's salts)(Thermo Fisher Scientific,目录号:61100061)
  10. 碳酸氢钠(NaHCO 3)(Carl Roth,目录号:6885.1)
  11. NEA(非必需氨基酸)(Biochrom,目录号:K 0293)
  12. 丙酮酸钠(EMD Millipore,目录号:106619)
  13. Tris-HCl(pH8.5)
  14. 胎牛血清(FBS)(Biowest,目录号:S181G)
  15. Lipofectamine ® 2000试剂(Thermo Fisher Scientific,目录号:11668027)
  16. Opti-MEM ®还原血清培养基(Thermo Fisher Scientific,目录号:31985062)
  17. 氯化钠(NaCl)(Carl Roth,目录号:9265.1)
  18. 氯化钾(KCl)(Carl Roth,目录号:5346.1)
  19. 葡萄糖(Sigma-Aldrich,目录号:D9434)
  20. 胰蛋白酶(1:250)粉末(Thermo Fisher Scientific,目录号:27250018)
  21. 乙二胺四乙酸(EDTA)(SERVA Electrophoresis,目录号:11280.01)
  22. 多聚甲醛(Carl Roth,目录号:0335.1)
  23. 10%成长媒体(见食谱)
  24. 3%多聚甲醛(见配方)
  25. 10 mM Tris HCl(pH 8.5)(见配方)
  26. Alsever's-Trypsin Versen(ATV-)解决方案(见配方)

注意:解决方案和媒体#6,11,14,15,16,21,22,23,24和26应保持在4°C。

设备

  1. T75烧瓶(例如,Corning,目录号:430725U)
  2. 孵化器(例如松下,三洋,型号:MCO-19AIC或Fisher Scientific,目录号:12826756)
  3. 荧光显微镜(例如,Nikon Instruments,型号:Eclipse Ti-S)
  4. 超高压汞灯电源(例如,尼康仪器,型号:C-SCH1)
  5. 光源(例如,尼康Instruments,型号:LH-M100C-1)
  6. Vortexer(例如,Fisher Scientific,目录号:S96461A)
  7. 层流罩(Thermo Fisher Scientific,Thermo Scientific TM,型号:Safe 2020 Class II,目录号:51026637)
  8. 1.5ml管的离心机(例如,Eppendorf,型号:5415D)
  9. 移液器控制器(例如,,pipetboy acu 2,VWR,目录号:37001-856)
  10. 纳米光度计(例如,VWR,IMPLEN,型号:P330,目录号:CA11027-294)

软件

  1. 计算机运行软件NIS-Elements V 4.00.01(Nikon,Düsseldorf,Germany)

程序

  1. 质粒的制备
    1. 用Tris-HCl pH8.5稀释表达推定的融合蛋白的质粒为200ng /μl(参见食谱)。包括表达自发荧光蛋白(例如,GFP或mCherry)的质粒作为标记以便于评估测定。这里,浓度为100ng /μl就足够了 注意:我们用于实验的表达质粒是基于pcDNA3,一种具有组织型转基因表达的哺乳动物表达载体,在人巨细胞病毒即时早期启动子/增强子复合物和新霉素抗性pEGFP-N1作为标记的控制下以便于评估测定(Schröter等,2015)

  2. 细胞的制备
    1. 在Eagle的最低必需培养基中补充10%FBS培养兔肾(RK13)细胞。将细胞用ATV(配方3)和种子〜1.8×10 5个细胞/孔胰蛋白酶消化至24孔细胞培养皿中。
    2. 第二天使用细胞进行转染。细胞应具有80-90%的汇合度。
      注意:针对最佳转染效率的细胞融合因细胞类型而异。对于RK13和Vero细胞,建议80-90%的汇合

  3. 转染
    1. 在单独的管中制备DNA混合物和Lipofectamine混合物。
      1. DNA混合物:
        使用200 ng的每种表达质粒,并稀释DNA混合物在50μlOpti-MEM。
      2. 脂质体混合物:
        在每孔50μlOpti-MEM中使用1μlLipofectamine 2000 TM。将Lipofectamine逐滴加入50μlOpti-MEM中,轻轻混合。在室温下孵育Lipofectamine混合物5分钟 注意:脂质体的最佳用量取决于细胞类型。对于RK13和Vero细胞,建议每24孔使用1μl。
    2. 将脂质体混合物滴加到DNA混合物中,轻轻敲打溶液并在室温(RT)下孵育20分钟(视频1)。
      注意:重要的是轻轻混合,彻底混合以形成DNA-脂质复合物。

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      视频1.教程:如何将Lipofectamine混合物逐滴加入DNA混合物
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    3. 从细胞中去除培养基,并将转染溶液逐滴加入细胞单层(视频2)。
      注意:为了允许转染溶液的相等分配,将其循环逐滴添加到单层上(视频2)。

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      视频2.教程:如何向细胞单层添加转染溶液
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    4. 向细胞中加入另外200μl的Opti-MEM以防止细胞干燥。
    5. 在37℃和5%CO 2下孵育细胞3小时。
    6. 3小时后,从细胞中取出转染溶液,并向细胞中加入补充有2%FBS的新鲜MEM,并在37℃和5%CO 2下孵育18-24小时。有关整个过程的概述,请参见图1.
      注意:为了获得可比较的结果,在固定细胞之前,在每个实验中使用完全相同的孵育时间。


      图1.转染方法的示意图

  4. 固定
    1. 准备3%的PFA(见食谱)。
    2. 使用摇杆在室温下,用约1ml PBS清洗培养基并洗涤细胞单层2分钟
    3. 取出PBS,每孔用200μl3%PFA固定细胞。在室温下固定细胞20分钟。
    4. 每孔用1ml PBS洗涤2×细胞。
    5. 每孔加入1 ml PBS,并将细胞储存在4°C。

  5. 融合活动的确定
    注意:为了量化融合活性,使用Nikon Eclipse Ti-S荧光显微镜和Nikon NIS-Elements成像软件测量具有三个或更多核的绿色合成细胞的数量和面积。
    1. 使用固定细胞通过荧光显微镜测定融合活性
    2. 打开NIS-Elements成像软件(Nikon)并选择相应类型的荧光和物镜(例如,FITC,10x)(参见图2红色框)。
    3. 选择测量手动测量区域(图2A,视频3)。
      注意:NIS-Elements支持的单位包括:像素,纳米,微米,毫米,厘米,分米,米,英寸和密耳。对于我们的测量,我们使用微米。


      图2. NIS Elements软件的示例视图。 A.选择手动测量; B.导出数据。
      注意:首先,测量屏幕右侧角落的合胞体积,因为每个合胞体都会在其右侧的站点上标有白色图标,这可能会妨碍右侧的合胞体的视图(参见图2B)。

    4. 每次在10个视场(〜5.5 mm 2 )中测量所有明显的合胞体,每次从相应图像的右上角开始。数据,包括每个合胞体的数量以及平均合胞体积将出现在右下角的表格中(见图2B绿色框,视频3)。

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      视频3.教程:如何使用NIS Elements软件测量合胞体系
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    5. 通过单击导出选项按钮将数据直接导出到Excel文件或文本文件(参见图3红色框)。 (选择是否将数据导出到Excel文件或文本文件。)您可以通过浏览按钮修改文件的名称和路径。
    6. 在三个不同的日子重复整个实验至少三次,以计算相对融合活性和相应的标准偏差(SD)。

数据分析

  1. 为了计算每个样本的总融合活性,对应于给定的孔,将合胞体数与10个视野的平均合胞体面积相乘。
    您计算了107个合胞体,平均合胞体积为7048μm,其融合活性为107×7,048.6 = 754,200.2。
  2. 通过比较三个独立实验中的每一个估计的融合活动,计算每个样本的总融合活性的标准偏差(SD)。
  3. 通过将其总融合活性与阳性对照(例如野生型gB,gH和gL)进行比较来确定不同样品的相对融合活性,其应当设定为100%。 br /> 例如,如果阳性对照的总融合活性为754,200.2,测试样品的融合活性为431,708.6,则测试样品的相对融合活性为(100/754,200.2)×431,708.6 = 57%(图3) 注意:使用Excel/Prism或类似的工具来促进计算和创建图形。
  4. 代表数据


    图3.融合活性为了测试融合活性,用编码野生型gB或突变型gB,gH,gL和gD以及EGFP的表达质粒共转染RK13细胞以促进测定通过荧光显微镜。用野生型gB与野生型gH,gL和gD组合的质粒进行测定为100%。用空载体pcDNA3作为阴性对照。转染后一天,测量含有三个或更多核的细胞的面积,并乘以合胞体数以确定融合活性。计算三个独立测定的相应标准偏差。

食谱

  1. 10%生长培养基(1L),pH7.2
    以下试剂应溶于双蒸水中至最终体积为1升:
    5.32克MEM鹰(汉克盐)
    4.76克MEM(厄尔盐)
    1.25g NaHCO 3
    10 ml NEA
    120毫克丙酮酸钠
  2. 10mM Tris-HCl,pH8.5
    准备2 M储备溶液(500 ml):
    121.14克Tris HCl
    用〜50 ml发烟HCl调节pH至8.5 稀释储备液1:20与ddH 2 O
  3. 阿尔卑斯胰蛋白酶维生素(ATV-)溶液,pH 7.2
    8.5克NaCl
    0.4克KCl
    1.0克右旋糖
    0.58g NaHCO 3
    0.5g胰蛋白酶1:250
    0.2g EDTA
    Ad 1 L ddH 2 O
  4. 3%PFA(100 ml)
    3克多聚甲醛
    100毫升PBS
    让PFA在65°C溶解

致谢

该协议是从以前的研究(Schroter等人,2015)中进行了修改和修改的。这项工作得到了DFG(ME 854/11)的资助。

参考文献

  1. Harrison,SC(2015)。  病毒膜融合 病毒学 479-480:498-507。
  2. Turner,A.,Bruun,B.,Minson,T.and Browne,H。(1998)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/9420303"target ="_ blank"> 1型单纯疱疹病毒的糖蛋白gB,gD和gHgL是必需的,足以介导Cos细胞转染系统中的膜融合。 em> 72(1):873-875。
  3. Muggeridge,MI(2000)。  细胞表征在转染细胞中由单纯疱疹病毒2糖蛋白gB,gD,gH和gL介导的融合。 Gen Virol 81(Pt 8):2017-2027。
  4. McShane,MP和Longnecker,R。(2005)。< a class ="ke-insertfile"href ="http://link.springer.com/protocol/10.1385%2F1-59259-848-X%3A187" target ="_ blank">使用无病毒细胞融合测定进行融合分析。方法Mol Biol 292:187-196。
  5. Schmidt,J.,Klupp,BG,Karger,A.and Mettenleiter,TC(1997)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/8985318"target ="_ blank">疱疹病毒的适应性:伪狂犬病病毒的糖蛋白D独立感染性。 71(1):17-24。
  6. Schröter,C.,Vallbracht,M.,Altenschmidt,J.,Kargoll,S.,Fuchs,W.,Klupp,BG and Mettenleiter,TC(2015)。< a class ="ke-insertfile"href = http://www.ncbi.nlm.nih.gov/pubmed/26656712"target ="_ blank">伪狂犬病病毒糖蛋白gB,gD和gH的突变功能上补偿gL的缺失。 J Virol 90(5):2264-2272。
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引用:Vallbracht, M., Schröter, C., Klupp, B. G. and Mettenleiter, T. C. (2017). Transient Transfection-based Fusion Assay for Viral Proteins. Bio-protocol 7(5): e2162. DOI: 10.21769/BioProtoc.2162.
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Gilles Michel
Millipore/Sigma
Why do you add fresh MEM supplemented with 2% FBS to the cells after transfection : why not 10% FBS ?. Is the goal to slow down the growth of the cells ?
10/13/2017 11:43:15 AM Reply
Melina Vallbracht
Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Germany

Thank you for your question. Yes, you are right: We reduce the FBS content to slow down the cell growth. If the RK13 cells that we use for transfection reach a confluency level which is too high, the cells would start being syncytial on its own. By reducing the FBS content we exclude that syncytia formation is induced by the confluency level. In addition, we control this by using a negative control (cells which are only transfected with the empty vector).

10/14/2017 10:04:50 AM