搜索

1 user has reported that he/she has successfully carried out the experiment using this protocol.
Alphavirus Purification Using Low-speed Spin Centrifugation
低速旋转离心纯化甲病毒   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Journal of Virology
Feb 2017

Abstract

Chemical and sedimentation procedures are used to purify virus particles. While these approaches are successful for wild-type viruses, they are often not feasible for purifying mutant viruses with assembly defects. We combined two published methods (Atasheva et al., 2013; Moller-Tank et al., 2013), to generate a protocol that uses low-speed centrifugation to purify both wildtype and mutant enveloped virus particles at high yield with minimal handling steps. This protocol has successfully been used to purify alphavirus particles for imaging and structural studies (Wang et al., 2015; Ramsey et al., 2017).

Keywords: Enveloped virus purification (包膜病毒纯化), Centrifugation (离心), Assembly mutants ( 组装突变体)

Background

Virus purification is traditionally based on chemical precipitation (e.g., PEG) or density gradient centrifugation. Centrifugation protocols involve pelleting particles at high speeds (> 100,000 x g) or through sedimentation matrices such as cesium, Nycodenz, Iodixanol, sucrose, glycerol, or tartrate. After sedimentation in the gradient, the purified virus sample usually requires additional steps to remove the gradient matrix, concentrate the purified particles, and buffer exchange into a stabilizing buffer for downstream applications. These approaches include dialysis, centrifugation through a centrifugal filter, or PEG precipitation. While these approaches will produce purified particles, there are several drawbacks: (1) overall yields can be low, (2) time required for the purification can extend to a week, (3) morphologically heterogeneous particles are not purified with equal efficiency, (4) particles can be damaged in the process, and (5) assembly mutants often do not survive the purification process making certain downstream analyses challenging.

The protocol described here uses a gentle approach to purify enveloped virus particles. We used minimal centrifugal force to reduce damage to particles which is observed as increased morphological heterogeneity in negative-stain transmission electron microscopy (TEM) or total loss of fragile particles by TEM or infectivity assays. In addition, we wanted to reduce the manipulation of purified virions post-purification. By merging two protocols from the literature (Atasheva et al., 2013; Moller-Tank et al., 2013), we are able to purify different Alphaviruses (Sindbis [Ramsey et al., 2017], Ross River [Wang et al., 2015], Chikungunya [Mukhopadhyay and Wang, unpublished data] and assembly mutants of each) via low-speed centrifugation (LSC). No additional steps for gradient matrix removal, sample concentration, or buffer exchange are necessary. We are also able to use this protocol to purify viruses from different cell lines (mammalian and arthropod). These purified particles have been used for cryo-EM, mass spectrometry, and protease cleavage studies.

Materials and Reagents

  1. Pipette tips
    Note: Filter tips are not necessary unless you routinely use when working with your virus of choice.
  2. 150 mm cell culture dishes (Greiner Bio One International, catalog number: 639160 , or equivalent)
  3. Sterile polypropylene 50 ml conical tubes (Corning, catalog number: 430921 , or equivalent)
  4. Serological pipets, 5 and 10 ml (DWK Life Sciences, KIMBLE, catalog numbers: 56900-5110 and 56900-10110 , or equivalent)
  5. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 , or equivalent)
  6. Cotton swabs (Ted Pella, catalog number: 80907 , or equivalent)
  7. pH test strips (Hach, catalog number: 2601300 )
  8. EM grids Formvar/Carbon 300 mesh (Ted Pella, catalog number: 01753-F )
  9. BHK-21 cells (ATCC, catalog number: CCL-10 )
  10. C6/36 cells (ATCC, catalog number: CRL-1660 )
  11. 1x sterile PBS (Corning, catalog number: 21-040-CM , or equivalent)
  12. Coomassie blue
  13. Page Ruler Prestained Protein Ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 22616 )
  14. MEM media (Corning, catalog number: 15-010-CV , or equivalent)
  15. 100x L-glutamine (Corning, catalog number: 25-005-CV , or equivalent)
  16. 100x MEM nonessential amino acids (Corning, catalog number: 25-025-Cl , or equivalent)
  17. 100x antibiotic-antimycotic (Corning, catalog number: 30-004-Cl , or equivalent)
  18. FBS (Corning, catalog number: 35-010-CV , or equivalent)
  19. Sterile virus production serum free media (VP-SFM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11681020 , or equivalent)
  20. HEPES (Fisher Scientific, catalog number: BP310-1 , or equivalent)
  21. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-10 , or equivalent)
  22. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS-100G , or equivalent)
  23. Hydrochloric acid (HCl)
  24. Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 , or equivalent)
  25. MEM complete media (see Recipes)
  26. Supplemented VP-SFM (see Recipes)
  27. HEPES-NaCl-EDTA (HNE) Resuspension buffer (see Recipes)
  28. 1% uranyl acetate (see Recipes)

Equipment

  1. Pipettes
  2. Tissue culture incubator, temperature and CO2 regulated (Thermo Fisher Scientific, Thermo ScientificTM, model: Forma 3130 , or equivalent)
  3. Refrigerated table-top centrifuge with fixed angle rotor (Eppendorf, model: 5804 R , with F34-6-38 rotor, or equivalent)
  4. Biosafety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II, Type A2 , catalog number: 1323TS, or equivalent)
  5. Adjustable tilt rocker (Reliable Scientific, model: 55 rocking shaker )

Procedure

  1. Seed 150 mm dishes with 8-9 x 106 BHK or C6/36 cells in 15 ml of complete MEM. For wild-type alphavirus purification, 2-3 150 mm dishes are routinely used in our lab.
  2. Grow BHK cells at 37 °C in 5% CO2 and C6/36 cells at 28 °C in 5% CO2 for 2-4 days until cells are > 95% confluent.
  3. Remove media using a serological pipette.
  4. Infect cells with alphavirus stock at a multiplicity of infection (MOI) = 5 plaque-forming units per cell. Make up the final volume to 5 ml with Supplemented VP-SFM.
  5. Rock for 1 h at speed control of 25 (on Reliable model 55 rocker) at room temperature so the virus has time to adhere and enter into cells. One-hour adsorption time is enough for both BHK and C6/36 cells.
  6. Remove media containing the virus.
  7. Add 5-10 ml PBS (equilibrated to room temperature) and tilt back and forth manually to wash the cells.
  8. Pipette off PBS.
  9. Repeat the PBS wash 2 more times. The goal is to remove as much of the MEM complete media as possible to avoid interference of residual FBS with the purification.
  10. Add 15 ml of room temperature Supplemented VP-SFM to each 150 mm plate.
  11. Incubate cells at an appropriate growth temperature. Media from BHK infected cells will be ready to harvest around 18-22 h post-infection. You should observe cytopathic effects and cell rounding, but minimal cell death (Figure 1). Prolonged incubation after the onset of the cytopathic effect will increase cell death and decrease virus yield. Infections in C6/36 cells incubate for 5 days but a cytopathic effect does not develop. After 2.5 days post-infection, add 5 ml of Supplemented VP-SFM media to the cells to supply additional nutrients. On day 5, collect the media and continue the next step.


    Figure 1. Display of CPE in BHK cells. Cells on the left (A) are uninfected and healthy whereas those on the right (B) have been infected with Sindbis virus for 20 h. Notice the cell rounding but yet minimal cell death. Images are at the same magnification (4,000x).

  12. Collect all the media in a 50 ml conical tube and clarify by centrifuging at 4,000 x g for 5 min, 4 °C. This step removes cell debris and dead cells. Transfer the supernatant into a new 50 ml conical tube.
  13. At this step, the sample can be kept at 4 °C. In our lab, we do not store the sample for more than 2 days. Do not freeze the sample as there is no serum present and the enveloped virus particles will be damaged.
  14. To purify the virus, centrifuge at 5,300 x g for 16 h at 15 °C. Use a fixed angle rotor in a refrigerated table-top centrifuge. Do not stop the centrifugation unless you are ready to continue with the subsequent steps immediately.
  15. Stop the centrifuge and, as you remove the tubes from the rotor, circle the position where the pellets have been collected on the side of the tube. The pellets are translucent at best, and are usually formed where the side and cone of the 50 ml conical tube meet (Figure 2). It can help to mark the wall of the tube for the following steps.


    Figure 2. Position of pelleted viruses in centrifugation tubes after LSC. When using a fixed-angle rotor, the pelleted virus will appear as a clear, transparent pellet (blue dots) on the upper wall of the conical tube.

  16. Carefully decant the supernatant without disturbing the pellet.
  17. Invert the conical tube onto a paper towel and let residual media drain down.
  18. Using a Kimwipe and a cotton swab, wipe dry the sides and bottom of the conical tube being careful not to disturb the pellet.
  19. Add 20-50 µl of the buffer of choice. PBS works well for biochemical assays and cryo-EM imaging but interferes with negative stain TEM. HNE buffer is good for both cryo-EM and negative stain TEM imaging. Volatile buffers such as ammonium acetate (100 mM, pH 7.5) are ideal for mass spectrometry applications.
  20. Store the resuspended virus sample at 4 °C; do not freeze. Note that over time particles may precipitate out of solution. We have stored viruses for up to 1 year and have not noticed degradation by SDS-PAGE or TEM analysis.

Data analysis

  1. SDS-PAGE
    Particle purity can be assessed by SDS-PAGE (Figure 3). For best separation, we run a 10% gel prepared under standard conditions. Gels are electrophoresed at 200 V for 40-50 min and can be stained with Coomassie blue or other conventional stains.


    Figure 3. Purity of virions prepared by the LSC protocol shown by SDS-PAGE. Sindbis virions from BHK-infected cells were purified using the LSC protocol. Three 150 mm dishes of cells were infected as described. LSC pellets were resuspended in 35 µl, and 2 µl of the resuspended pellet was loaded on the SDS-PAGE gel. The ladder is PageRuler Prestained Protein Ladder from Invitrogen. The bands seen on the gel correspond to the outer glycoproteins and the inner capsid protein and are labeled accordingly. Note, only viral proteins are observed.

  2. TEM
    Particle morphology is assessed by transmission electron microscopy (TEM) (Figure 4). Resuspended virus samples (3-5 µl) are applied to a carbon- and formvar-coated copper grid for 1 min and then blotted dry by holding a Kimwipe to the side of the grid. When blotting, place the paper at the edge of the grid in a perpendicular orientation. Thinner paper (or Kimwipes) wicks more completely than the thicker materials. Before the grid dries out (within seconds), add 5 µl of 1% uranyl acetate for 5 min. Uranyl acetate is removed by blotting with the side of the grid against a Kimwipe. Samples prepared this way can be imaged by any voltage TEM.


    Figure 4. Purification of wild-type and assembly mutant virus. Sindbis virions (wild-type and an assembly mutant) from both BHK-infected and C6/36-infected cells were purified using two protocols and then imaged by TEM. The top row shows the virion purification using three sequential steps: pelleting through a sucrose cushion, applying the pellet on a sucrose gradient, and buffer exchange and concentration of the viral band using centrifugal filtration. The bottom row shows the virus samples from the one step virion purification using the LSC protocol. In addition to having more particles on the grid, the LSC purified particles show fragile particles as indicated by the dent in the middle of the particle (arrow). These particles are most likely degraded or further damaged during the harsher purification methods. As a result, the diversity of assembly mutants is not represented in the upper panels. Scale bar (shown bottom right image) is 200 nm and is applicable to all EM images.

Notes

  1. General notes
    1. Each virus has a different infection MOI and harvest time. Additionally, different cell lines will also influence the optimal conditions for virus production. Performing a growth curve using your virus and host cell of interest is a good starting point for establishing purification harvest times.
    2. After harvesting media (Step 12), if the media appears slightly acidic after harvesting the cells (Step 12) (observed by orange tint or measuring with pH paper), add sterile 1 M HEPES buffer, pH 7.5 to a final concentration of 5-10 mM to help to maintain the pH between 7-7.5.
    3. When we want to use one purified virus preparation for multiple downstream analyses, we resuspend the low-speed centrifuged pellet in a larger volume (up to 500 µl) and then take portions of this sample for further applications.
    4. In our laboratory, this protocol has been used for alphaviruses propagated in both BHK and C6/36 cells. We have not tried purifying viruses from other cell lines.

Recipes

  1. MEM complete media
    500 ml MEM
    5 ml 100x L-glutamine
    5 ml 100x nonessential amino acids
    5 ml 100x antibiotic-antimycotic
    50 ml FBS
    Add all components to 500 ml bottle of MEM; the final volume is 565 ml
    Store at 4 °C up to 6 months
  2. Supplemented VP-SFM
    1 L VP-SFM
    10 ml 100x L-glutamine
    10 ml 100x nonessential amino acids
    10 ml 100x antibiotic-antimycotic
    Store at 4 °C up to 6 months
    Add all components to 1 L bottle of VP-SFM; the final volume is 1,030 ml
  3. HEPES-NaCl-EDTA (HNE) Resuspension buffer (pH = 7.5)
    20 mM HEPES
    150 mM NaCl
    0.1 mM EDTA
    Adjust pH using HCl
    Store at room temperature
  4. 1% uranyl acetate
    Make a 10% solution (Vf = 1 ml) by adding 0.1 g uranyl acetate to 1 ml of MilliQ water. Invert to mix. The solution will be yellow
    To make 1% solution (Vf = 1 ml), take 100 µl of 10% solution and add 900 µl of MilliQ water. Invert to mix
    Store in the dark at room temperature
    Note: Uranyl acetate waste should be disposed of according to the institutional policy.

Acknowledgments

This protocol was adapted from the previous publications Atasheva et al., 2013 and Moller-Tank et al., 2013. It has been used in Wang et al., 2015 and Ramsey et al., 2017. We acknowledge members of the Mukhopadhyay laboratory who shared purified alphavirus virions for our analyses. Funding for this work was from the National Science Foundation and Indiana University Office of the Vice Provost for Research. The authors declare no conflicts of interest or competing interests.

References

  1. Atasheva, S., Kim, D. Y., Akhrymuk, M., Morgan, D. G., Frolova, E. I. and Frolov, I. (2013). Pseudoinfectious Venezuelan equine encephalitis virus: a new means of alphavirus attenuation. J Virol 87(4): 2023-2035.
  2. Moller-Tank, S., Kondratowicz, A. S., Davey, R. A., Rennert, P. D. and Maury, W. (2013). Role of the phosphatidylserine receptor TIM-1 in enveloped-virus entry. J Virol 87(15): 8327-8341.
  3. Ramsey, J., Renzi, E. C., Arnold, R. J., Trinidad, J. C. and Mukhopadhyay, S. (2017). Palmitoylation of Sindbis virus TF protein regulates its plasma membrane localization and subsequent incorporation into virions. J Virol 91(3).
  4. Wang J. C., Chen, C., Rayaprolu, V., Mukhopadhyay, S. and Zlotnick A. (2015). Self-assembly of an alphavirus core-like particle is distinguished by strong intersubunit association energy and structural defects. ACS Nano 9(9): 8898-8906.

简介

化学和沉淀过程用于纯化病毒颗粒。 虽然这些方法对于野生型病毒是成功的,但它们通常不能用于纯化具有装配缺陷的突变病毒。 我们结合了两种公开的方法(Atasheva等人,2013; Moller-Tank等人,2013),以生成使用低速离心纯化两种方法的方案 野生型和突变体包膜病毒颗粒,产量高,处理步骤最少。 该方案已成功用于纯化甲病毒颗粒用于成像和结构研究(Wang等人,2015; Ramsey等人,2017)。

【背景】传统上,病毒纯化基于化学沉淀(例如PEG)或密度梯度离心。离心方案包括以高速(>100,000μgx g)或通过沉淀基质如铯,Nycodenz,碘克沙醇,蔗糖,甘油或酒石酸丸粒化颗粒。在梯度沉降之后,纯化的病毒样品通常需要额外的步骤来除去梯度基质,浓缩纯化的颗粒,并且缓冲液交换成用于下游应用的稳定缓冲液。这些方法包括透析,通过离心过滤器离心或PEG沉淀。虽然这些方法会产生纯化的颗粒,但有几个缺点:(1)总产量可能很低,(2)纯化所需的时间可能延长到一周,(3)形态上不均匀的颗粒不能以相同的效率纯化,( 4)过程中可能会损坏颗粒,以及(5)组装突变体常常不能在净化过程中存活,使得某些下游分析具有挑战性。

这里描述的协议使用温和的方法来净化包膜病毒颗粒。我们使用最小的离心力来减少对粒子的损伤,这是由于负染透射电子显微镜(TEM)中增加的形态异质性或通过TEM或传染性测定法测定的易碎粒子的全部损失而观察到的。另外,我们希望减少纯化后纯化病毒体的操作。通过合并文献中的两个方案(Atasheva等人,2013; Moller-Tank等人,2013),我们能够纯化不同的α病毒 (Sindbis [Ramsey等人,2017],Ross River [Wang等人,2015],Chikungunya [Mukhopadhyay和Wang,未发表的资料]和汇编各自的突变体)通过低速离心(LSC)进行。不需要额外的梯度矩阵去除步骤,样品浓度或缓冲液交换步骤。我们也可以使用这个协议从不同细胞系(哺乳动物和节肢动物)中纯化病毒。这些纯化的颗粒已用于冷冻电镜,质谱和蛋白酶切割研究。

关键字:包膜病毒纯化, 离心, 组装突变体

材料和试剂

  1. 移液器吸头
    注意:除非您在处理您选择的病毒时经常使用,否则过滤器提示不是必需的。

  2. 150毫米细胞培养皿(Greiner Bio One International,目录号:639160,或同等学历)
  3. 无菌聚丙烯50毫升锥形管(Corning,目录号:430921,或同等产品)
  4. 血清学移液管,5和10毫升(DWK生命科学公司,KIMBLE,目录号:56900-5110和56900-10110,或同等学历)
  5. Kimwipes(KCWW,Kimberly-Clark,目录号:34155,或同等学历)
  6. 棉签(Ted Pella,产品目录号:80907,或同等产品)
  7. pH试纸(Hach,目录号:2601300)
  8. EM网格Formvar / Carbon 300目(Ted Pella,目录号:01753-F)
  9. BHK-21细胞(ATCC,目录号:CCL-10)
  10. C6 / 36细胞(ATCC,目录号:CRL-1660)
  11. 1x无菌PBS(Corning,目录号:21-040-CM或同等产品)
  12. 考马斯蓝
  13. Page Ruler Prestained Protein Ladder(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:22616)
  14. MEM培养基(Corning,产品目录号:15-010-CV或同等产品)
  15. 100x L-谷氨酰胺(康宁公司,产品目录号:25-005-CV或同等产品)
  16. 100x MEM非必需氨基酸(Corning,目录号:25-025-Cl或同等产品)
  17. 100x抗生素 - 抗霉菌剂(康宁,目录号:30-004-Cl或同等产品)
  18. FBS(Corning,产品目录号:35-010-CV或同等产品)
  19. 无菌病毒产生无血清培养基(VP-SFM)(Thermo Fisher Scientific,Gibco TM,目录号:11681020或等同物)
  20. HEPES(Fisher Scientific,产品目录号:BP310-1,或同等产品)
  21. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358-10,或同等产品)
  22. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS-100G,或同等产品)
  23. 盐酸(HCl)
  24. 乙酸铀酰(电子显微镜科学,目录号:22400,或同等学历)
  25. MEM完整媒体(见食谱)
  26. 补充的VP-SFM(见食谱)
  27. HES EPES-N aCl-E DTA(HNE)重悬缓冲液(参见食谱)
  28. 1%醋酸铀(见食谱)

设备

  1. 移液器
  2. 组织培养孵育器,温度和CO 2调节(赛默飞世尔科技,Thermo Scientific TM,型号:Forma 3130,或同等产品)
  3. 具有固定角度转子的冷冻台式离心机(Eppendorf,型号:5804 R,带有F34-6-38转子或同等产品)
  4. 生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300系列Class II,Type A2,目录号:1323TS或同等产品)
  5. 可调倾斜摇杆(可靠的科学,型号:摇摆摇床)

程序

  1. 在15ml完全MEM中种植具有8-9×10 6 BHK或C6 / 36细胞的150mm培养皿。对于野生型α病毒纯化,在我们的实验室中常常使用2-3个150毫米培养皿。
  2. 在37℃,5%CO 2和C6 / 36细胞中于28℃,在5%CO 2 2中培养BHK细胞2-4天,直至细胞> 95%融合。
  3. 使用血清移液管移除介质。
  4. 以感染复数(MOI)=每个细胞5个噬菌斑形成单位感染具有α病毒原种的细胞。
    补充VP-SFM使最终体积达到5毫升
  5. 在室温下摇动速度控制在25(在Reliable 55型摇床上)1小时,以便病毒有时间粘附并进入细胞。
    一小时吸附时间对BHK和C6 / 36细胞都是足够的
  6. 移除含有病毒的媒体。
  7. 加入5-10 ml PBS(平衡至室温)并手动来回倾斜以清洗细胞。
  8. 吸取PBS。
  9. 重复PBS洗涤2次。我们的目标是尽可能多地去除MEM完整的培养基,以避免残留FBS与纯化的干扰。

  10. 在每个150毫米的平板上加入15毫升室温辅助VP-SFM
  11. 在适当的生长温度下孵育细胞。来自BHK感染细胞的培养基将在感染后18-22小时准备好收获。您应该观察到细胞病变效应和细胞四舍五入,但最小的细胞死亡(图1)。细胞病变效应开始后的长时间孵育会增加细胞死亡并降低病毒产量。 C6 / 36细胞中的感染孵育5天,但细胞病变效应不发展。感染后2.5天后,向细胞中加入5毫升补充的VP-SFM培养基以提供额外的营养。在第5天,收集媒体并继续下一步。


    图1.在BHK单元中显示CPE。 左侧的细胞(A)未感染且健康,而右侧的细胞(B)已经感染辛德比斯病毒20小时。请注意细胞四舍五入,但最小的细胞死亡。图像的放大倍数相同(4,000倍)。

  12. 将所有培养基收集在50毫升锥形管中,并通过在4,000×g <4:5的条件下离心5分钟进行澄清。这一步消除细胞碎片和死细胞。将上清液转移到新的50 ml锥形管中。
  13. 在这一步,样品可以保持在4°C。在我们的实验室中,我们不会将样品存储超过2天。
    不要冻结样品,因为没有血清存在,并且有包膜的病毒颗粒会被损坏。
  14. 为了纯化病毒,在15℃下以5,300×g 离心16小时。在冷藏台式离心机中使用固定角度的转子。
    不要停止离心,除非您准备好立即继续后续步骤。
  15. 停止离心机,当您从转子上取下管子时,将管子一侧的颗粒收集位置圈起来。颗粒最好是半透明的,通常在50ml圆锥形管的侧面和圆锥体相遇处形成(图2)。
    这可以帮助标记管的壁以进行以下步骤

    图2. LSC后离心管中沉淀病毒的位置。 使用固定角度的转子时,沉淀的病毒在圆锥形管上壁显示为透明,透明的颗粒(蓝色圆点)。

  16. 仔细倾析上清液而不会干扰颗粒。

  17. 。将锥形管倒置在纸巾上,让剩余的介质排出。
  18. 使用Kimwipe和棉签,擦干圆锥管的侧面和底部,注意不要弄乱颗粒。
  19. 加入20-50μl的选择的缓冲液。 PBS适用于生化分析和冷冻电镜成像,但会干扰阴性染色透射电镜。 HNE缓冲液对于冷冻EM和阴性染色TEM成像都是有利的。挥发性缓冲液如醋酸铵(100 mM,pH 7.5)是质谱应用的理想选择。
  20. 将重新悬浮的病毒样品保存在4°C;不要冻结。请注意,随着时间的推移,颗粒可能从溶液中沉淀出我们已经存储了长达1年的病毒,并没有发现通过SDS-PAGE或TEM分析的降解。

数据分析

  1. SDS-PAGE
    颗粒纯度可以通过SDS-PAGE评估(图3)。为了获得最佳分离效果,我们运行标准条件下制备的10%凝胶。凝胶在200V电泳40-50分钟,可用考马斯蓝或其他常规染色剂染色。


    图3.通过SDS-PAGE显示的LSC方案制备的病毒粒子的纯度。使用LSC方案纯化来自BHK感染的细胞的辛德毕斯病毒颗粒。如所述感染三个150mm细胞细胞。将LSC沉淀物重新悬浮于35μl中,并将2μl重悬浮的沉淀物加载到SDS-PAGE凝胶上。梯子是来自Invitrogen的PageRuler Prestained Protein Ladder。在凝胶上看到的条带对应于外部糖蛋白和内部衣壳蛋白,并相应地进行标记。请注意,只有病毒蛋白被观察到。

  2. TEM
    透射电子显微镜(TEM)评估颗粒形态(图4)。将悬浮的病毒样品(3-5μl)施加到碳涂层和formvar涂覆的铜网格上1分钟,然后通过将Kimwipe固定在格栅的侧面进行干燥。在吸墨时,将纸张垂直放置在网格边缘。较薄的纸(或Kimwipes)比较厚的材料更彻底地吸湿。在电网干燥之前(几秒钟内),加入5μl1%醋酸铀酰5分钟。通过将格栅侧面对Kimwipe进行印迹来除去乙酸双氧铀。用这种方法制备的样品可以通过任何电压TEM成像。


    图4.纯化野生型和装配突变病毒。来自BHK感染和C6 / 36感染的细胞的辛德毕斯病毒体(野生型和装配突变体)使用两种方案纯化,然后通过TEM成像。最上面一行显示了病毒粒子纯化,使用三个连续的步骤:通过蔗糖垫片造粒,在蔗糖梯度上施加沉淀,以及使用离心过滤进行缓冲液交换和病毒带的浓缩。最下面一行显示了使用LSC方案进行的一步病毒体纯化的病毒样品。除了在网格上具有更多的粒子之外,LSC纯化的粒子显示了易碎的粒子,如粒子中间的凹痕所示(箭头)。在严酷的纯化方法中,这些颗粒很可能会降解或进一步受损。结果,组装突变体的多样性未在上图中示出。比例尺(右下图所示)为200 nm,适用于所有EM图像。

笔记

  1. 一般注意事项
    1. 每种病毒都有不同的感染MOI和收获时间。此外,不同的细胞系也会影响病毒生产的最佳条件。使用感兴趣的病毒和宿主细胞进行生长曲线是建立纯化收获时间的良好起点。
    2. 收获培养基后(步骤12),如果培养基收获细胞后出现微酸性(步骤12)(用橙色或用pH纸测量),加入无菌1M HEPES缓冲液(pH7.5)至终浓度为5% 10 mM有助于维持pH值在7-7.5之间。
    3. 当我们想要使用一种纯化的病毒制剂进行多次下游分析时,我们将较低体积的离心沉淀物(最多500μl)重新悬浮,然后取出部分样品进行进一步应用。
    4. 在我们的实验室中,该方案已用于在BHK和C6 / 36细胞中繁殖的甲病毒。我们还没有尝试从其他细胞系中纯化病毒。

食谱

  1. MEM完整媒体
    500毫升MEM
    5毫升100倍L-谷氨酰胺
    5毫升100倍非必需氨基酸
    5毫升100x抗生素 - 抗真菌剂
    50毫升FBS
    将所有组分加入500毫升MEM培养瓶中;最终的体积是565毫升

    在4°C储存至6个月
  2. 补充VP-SFM
    1 L VP-SFM
    10毫升100倍L-谷氨酰胺
    10毫升100倍非必需氨基酸
    10毫升100x抗生素 - 抗真菌剂

    在4°C储存至6个月 将所有组分添加到1L VP-SFM瓶中;最终成交量为1,030毫升
  3. EPES-N aCl-E DTA(HNE)再悬浮缓冲液(pH = 7.5)
    20mMHEPES
    150毫摩尔氯化钠 0.1毫摩尔DTA
    用HCl调节pH值
    在室温下储存
  4. 1%醋酸铀酰
    通过将0.1g乙酸铀酰加入到1ml MilliQ水中来制备10%溶液(Vf = 1ml)。颠倒混合。解决方案将是黄色
    要制备1%溶液(Vf = 1 ml),取100μl10%溶液并加入900μlMilliQ水。颠倒混合

    在室温下避光存放 注意:乙酸铀酰废物应根据机构政策处理。

致谢

该协议改编自以前的出版物Atasheva et al。,2013和Moller-Tank et。,2013。它已被用于Wang et al。 ,2015年和Ramsey 等人,2017年。我们承认Mukhopadhyay实验室的成员为我们的分析共享纯化的α病毒毒粒。这项工作的资金来自美国国家科学基金会和印第安纳大学研究副校长办公室。作者声明不存在利益冲突或利益冲突。

参考

  1. Atasheva,S.,Kim,D.Y.,Akhrymuk,M.,Morgan,D.G.,Frolova,E.I和Frolov,I。(2013)。 假感染委内瑞拉马脑炎病毒:α病毒减毒的新手段。 Virol 87(4):2023-2035。
  2. Moller-Tank,S.,Kondratowicz,A. S.,Davey,R. A.,Rennert,P.D。和Maury,W。(2013)。 磷脂酰丝氨酸受体TIM-1在包膜病毒进入中的作用 J Virol 87(15):8327-8341。
  3. Ramsey,J.,Renzi,E.C.,Arnold,R.J.,Trinidad,J.C。和Mukhopadhyay,S。(2017)。 辛德毕斯病毒TF蛋白的棕榈酰化调节其质膜定位和随后掺入病毒粒子。 J Virol 91(3)。
  4. Wang J. C.,Chen,C.,Rayaprolu,V.,Mukhopadhyay,S.和Zlotnick A.(2015)。 α病毒核心样颗粒的自组装通过强烈的亚基间缔合能量和结构缺陷来区分。 ACS Nano 9(9):8898-8906。
  • English
  • 中文翻译
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: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rayaprolu, V., Ramsey, J., Wang, J. C. and Mukhopadhyay, S. (2018). Alphavirus Purification Using Low-speed Spin Centrifugation. Bio-protocol 8(6): e2772. DOI: 10.21769/BioProtoc.2772.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。