欢迎您, 登录 | 注册

首页 | English

X
加载中

This protocol was designed for large-scale purification of herpesvirus particles by cell culture. Virions and capsids are isolated from extracellular culture media and cell nuclei, respectively. Purity and concentration of the purified samples are usually sufficient for structural studies with cryo electron microscopy and cryo electron tomography. The protocol should also be generally suitable for purifying herpesvirus virions and capsids for other types of studies.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Purification of Herpesvirus Virions and Capsids
疱疹病毒和病毒衣壳的纯化

微生物学 > 微生物细胞生物学 > 细胞分离和培养
作者: Xinghong Dai
Xinghong DaiAffiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, USA
Bio-protocol author page: a1532
 and Z. Hong Zhou
Z. Hong ZhouAffiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, USA
For correspondence: hong.zhou@ucla.edu
Bio-protocol author page: a1533
Vol 4, Iss 15, 8/5/2014, 3825 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1193

[Abstract] This protocol was designed for large-scale purification of herpesvirus particles by cell culture. Virions and capsids are isolated from extracellular culture media and cell nuclei, respectively. Purity and concentration of the purified samples are usually sufficient for structural studies with cryo electron microscopy and cryo electron tomography. The protocol should also be generally suitable for purifying herpesvirus virions and capsids for other types of studies.
Keywords: Virus purification(病毒纯化), Herpesvirus(疱疹病毒), Density gradient centrifuge(密度梯度离心)

[Abstract]

Materials and Reagents

  1. Herpesvirus infected cells (e.g., Vero cells for herpes simplex virus culture, MRC5 cells for human cytomegalovirus culture)
  2. PBS (pH 7.4)
  3. Sucrose
  4. NP-40
  5. Dry ice
  6. Dulbecco’s modified eagle medium (DMEM)
  7. Fetal bovine serum (FBS)
  8. Herpesvirus culture media (see Recipes)

Equipment

  1. Laminar flow hood
  2. Centrifuge with cool function
  3. Ultracentrifuge
  4. Ultra-Clear 38.5 ml tubes (fit in SW28 rotor) (Beckman Coulter, catalog number: 344058 )
  5. Ultra-Clear 13.2 ml tubes (fit in SW41 rotor) (Beckman Coulter, catalog number: 344059 )
  6. KimwipesTM tissue paper
  7. Tweezers
  8. Gradient master (Biocomp Instruments, catalog number: 107-201M )
  9. Flashlight
  10. Vortex
  11. 37 °C water bath
  12. Syringe with 23 gauge hypodermic needle

Procedure

  1. Purify virion from herpesvirus culture media
    1. When the virus culture is ready for harvest, collect the culture supernatant in a laminar flow hood. We usually work with 20-30 175 cm2 flasks of cell culture for each batch of purification.
      Note: From this step on, always keep sample on ice; all centrifuge should be done at 4 °C.
    2. Spin 15 min at ~10,000 x g to remove cell debris (8,000 rpm with Beckman Coulter JA-14 rotor). For large volume (e.g., over 200 ml for each bottle), may need to spin longer or repeat once to completely clarify the media.
    3. Collect the supernatant from last step, ultracentrifuge at ~80,000 x g for 1 h to pellet the virion (21,000 rpm with SW28 rotor).
      Note: Even for smaller virus (e.g., Dengue virus around 50 nm), ultracentrifuge at 80,000 x g for 1 h is enough to pellet down most of the virus particles. Centrifuge at higher speed or for longer time may cause structural damage to some of the virus particles, or make the pellet hard to be resuspended.
    4. Dump the supernatant. Use a KimwipesTM tissue paper winded around a pair of tweezers to completely wipe out media on the inner wall of the tube. Add 100 µl PBS for each tube, incubate on ice overnight or for at least 2 h, then resuspend the pellet by pipetting.
      Note: Soaking the pellet in solution for hours helps to loosen the pellet and makes it easier to be completely resuspended.
    5. Make a continuous density gradient in SW41 centrifuge tube with ~5 ml each 15% and 50% (w/v) sucrose solution in PBS, using the Gradient Master machine. Load the resuspension on top of the density gradient. Use another tube with sucrose solutions for balance.
      Note: It’s better to load less than 2 ml (at most 3 ml) sample for each density gradient tube. Too large volume of sample may lead to a smeared band in the next steps.
    6. Spin at 80,000 x g for 1 h (21,000 rpm with SW41 rotor).  
    7. In a dark room, shine a flashlight on top of the density gradient tube to clearly see the virus band and then mark the position. A good virus band should be a shiny, milky line with clear-cut interface. For herpesvirus virion purification, sometimes two bands can be identified. Normally, the upper band contains more virions with empty A- or B-capsids, while the lower band contains more dense bodies. But majority of particles in both bands are virions with DNA-filled C-capsids.
    8. In a laminar flow hood, remove the gradient solution above the virus band with 1 ml pipette, then collect the band with new pipette. Try to keep the volume as small as possible to avoid impurities above and below the band.  For low concentration samples, if no band identified, aliquot the gradient into 2 ml each.
    9. Dilute the virus band (or each 2 ml aliquot) with PBS in SW41 tube to the full volume of the tube. Spin at 80,000 x g for 1 h to pellet the purified virus.
    10. Dump the supernatant. Use KimwipesTM tissue paper to clean inner wall of the tube as in step A4. Add 10-30 μl (depending on size of the pellet and desired final concentration) PBS, incubate on ice for at least 2 h or overnight, and then resuspend the pellet by pipetting.
    11. If no band identified and aliquots collected in step A7, check each sample with negative-staining Electron Microscopy to figure out which one has virus particle.
      Note: For herpesvirus, it’s hard to distinguish virion from dense body with negative staining EM. Mix 2 μl of the sample with equal volume of 1% NP-40 in PBS to partially dissolve the virus envelope before negative staining, then viral capsid in the virion will be stained to have a spiky appearance which is easy to identify.

  2. Purify viral capsid from herpesvirus infected cells
    1. Harvest herpesvirus-infected cells when reaching 90% cytopathic effect. We normally start with 15-20 175 cm2 flasks of cell culture.
    2. Pellet cells by centrifuge at 1,000 x g at 4 °C for 10 min.  Remove the supernatant.
    3. Wash the pellet with 30 ml pre-chilled PBS and centrifuge again at 1,000 x g at 4 °C for 10 min. Remove the supernatant.
    4. Loosen the pellet by vortexing at half maximum speed for 5 sec. Resuspend the pellet with 30 ml 0.5% (w/v) NP-40 in PBS by pipetting and incubate the mixture on ice for 5 min.
    5. Centrifuge at 1,000 x g at 4 °C for 10 min. Remove the supernatant.
    6. Resuspend the pellet (containing cell nuclei) in 30 ml PBS and lyse by three cycles of freezing (dry ice or -80 °C, 10 min), thawing (37 °C water bath, 3-4 min), and 1 min vortexing.
    7. Keep the lysate on ice. Pass the lysate through a 23 gauge hypodermic needle for at least 20 times.
    8. Add 10% (w/v) NP-40 in PBS to the lysate to make final concentration of 2% NP-40. Incubate at 4 °C overnight.
    9. Centrifuge at 1,500 x g at 4 °C for 5 min to remove large debris.
    10. Take 5 ml 25% sucrose (w/v in PBS) in SW28 tube, and then inject 5 ml 50% sucrose (w/v in PBS) with a syringe to the bottom of the tube, to make a double-layer sucrose cushion. Gently load the lysate (supernatant containing capsids) on the cushion with pipette and centrifuge at 80,000 x g for 1 h.
    11. Collect the band at interface of the two sucrose layers. Dilute with PBS for two to three times, load on a continuous 15% to 50% (w/v in PBS) sucrose density gradient. Load less than 2 ml sample for each gradient tube; use multiple tubes if have more sample.  Centrifuge at 80,000 x g for 1 h.
    12. Collect the band of capsid, dilute with PBS in SW41 tube to full volume of the tube, centrifuge at 80,000 x g for 1 h to pellet the capsid. For herpesvirus capsid purification, sometimes three bands can be identified. They contain A-, B-, and C-capsids for the top-, middle-, and bottom-band respectively.  
    13. Dump the supernatant, wipe out all liquid on the inner wall of the tube with KimwipesTM tissue paper and tweezers. Add 10-30 μl PBS, incubate on ice for at least 2 h or overnight, and then resuspend the pellet by pipetting.
    14. Check purity and concentration of the sample by negative staining electron microscopy or cryo electron microscopy.

Recipes

  1. Herpesvirus culture media
    Dulbecco’s modified eagle medium with 10% fetal bovine serum

Acknowledgments

This protocol was adapted from our previously published paper Dai et al. (2013). Citation of this original paper is also encouraged when referencing the protocol. This work was supported by a grant from the National Institutes of Health (NIH) to Z. Hong Zhou (AI069015).

References

  1. Dai, X., Yu, X., Gong, H., Jiang, X., Abenes, G., Liu, H., Shivakoti, S., Britt, W. J., Zhu, H., Liu, F. and Zhou, Z. H. (2013). The smallest capsid protein mediates binding of the essential tegument protein pp150 to stabilize DNA-containing capsids in human cytomegalovirus. PLoS Pathog 9(8): e1003525.

材料和试剂

  1. 疱疹病毒感染的细胞(例如,用于单纯疱疹病毒培养的Vero细胞,用于人巨细胞病毒培养的MRC5细胞)
  2. PBS(pH 7.4)
  3. 蔗糖
  4. NP-40
  5. 干冰
  6. Dulbecco改良的Eagle培养基(DMEM)
  7. 胎牛血清(FBS)
  8. 疱疹病毒培养基(见配方)

设备

  1. 层流罩
  2. 带冷功能离心器
  3. 超速离心机
  4. 超透明38.5ml管(装入SW28转子)(Beckman Coulter,目录号:344058)
  5. 超透明13.2ml管(装配在SW41转子中)(Beckman Coulter,目录号:344059)
  6. Kimwipes TM 薄纸
  7. 镊子
  8. 梯度master(Biocomp Instruments,目录号:107-201M)
  9. 手电筒
  10. 涡流
  11. 37°C水浴
  12. 带23号皮下注射针的注射器

程序

  1. 从疱疹病毒培养基中纯化病毒体
    1. 当病毒培养物准备收获时,收集培养物 上清液在层流罩中。 我们通常使用20-30 175cm 2细胞培养瓶进行每批纯化。
      注意:从这一步开始,始终将样品保持在冰上; 所有离心机应在4℃下进行。
    2. 以〜10,000×g离心15分钟以除去细胞碎片(8,000rpm, Beckman Coulter JA-14转子)。 对于大容量(例如,超过200毫升 每个瓶子),可能需要旋转更长或重复一次完全 澄清媒体。
    3. 收集上一步的上清液, 在〜80,000×g下超速离心1小时以沉淀病毒颗粒(21,000rpm 与SW28转子)。
      注意:即使是较小的病毒(例如,登革病毒 约50nm),以80,000xg超速离心1小时就足以沉淀   下来大多数病毒颗粒。 离心机以更高的速度或 较长的时间可能会对一些病毒颗粒造成结构性损伤, 或使颗粒难以重悬。
    4. 转储 上清液。使用Kimwipes TM 薄纸卷绕在一对 镊子彻底擦拭管内壁上的介质。加  每个管100μlPBS,在冰上孵育过夜或至少2 h,然后通过吸移重悬浮沉淀。
      注意:将沉淀在溶液中浸泡数小时有助于松散沉淀,并使其更容易完全重悬。
    5. 在SW41离心管中用〜5 ml制备连续密度梯度 每个15%和50%(w/v)蔗糖的PBS溶液 主机。将重悬浮液加载到密度梯度的顶部。 使用另一管与蔗糖溶液平衡。
      注意:这是 更好地为每种密度加载小于2ml(至多3ml)的样品 梯度管。样品体积过大可能导致模糊带 下一步。
    6. 在80,000×g下旋转1小时(使用SW41转子,21,000rpm)。  
    7. 在黑暗的房间里,在密度梯度管的顶部照射手电筒   清楚地看到病毒带,然后标记位置。 一个好的病毒 带应该是有光泽,乳白色的线,具有清晰的界面。 对于 疱疹病毒病毒体纯化,有时可以鉴定两条带。 通常,上带包含更多具有空A-或的病毒粒子 B衣壳,而下带包含更致密的体。 但多数   的两个条带中的颗粒是具有DNA填充的C衣壳的病毒体。
    8. 在层流罩中,去除病毒上方的梯度溶液 带用1ml移液管,然后用新的移液管收集带。 尝试 保持体积尽可能小,以避免上下的杂质   乐队。 对于低浓度样品,如果没有鉴定到条带, 等分梯度每2毫升。
    9. 稀释病毒带(或   每2ml等分)用PBS在SW41管中至全部体积 管。 以80,000×g离心1小时以沉淀纯化的病毒。
    10. 倾倒上清液。 使用Kimwipes TM 棉纸清洁内壁 如在步骤A4中。 加入10-30μl(取决于沉淀的大小   和期望的最终浓度)PBS,在冰上孵育至少2小时 或过夜,然后通过吸移重悬浮沉淀。
    11. 如果 在步骤A7中没有收集到带和等分试样,检查每个样品 用负染色电子显微镜来确定哪一个 病毒颗粒。
      注意:对于疱疹病毒,很难区分 病毒体从致密体与阴性染色EM。 混合2μl的样品   用等体积的1%NP-40的PBS溶液以部分溶解病毒 包膜前阴性染色,则病毒体中的病毒衣壳 被染色以具有容易识别的尖锐外观。

  2. 从疱疹病毒感染的细胞中纯化病毒衣壳
    1. 收获疱疹病毒感染细胞达到90%的细胞病变效应。   我们通常从15-20 175cm 2细胞培养瓶开始。
    2. 通过在4℃下以1,000×g离心10分钟离心沉淀细胞。 取出上清液。
    3. 用30ml预冷的PBS洗涤沉淀,并在1,000xg下再次在4℃离心10分钟。 取出上清液。
    4. 通过以半最大速度涡旋5秒钟来松开沉淀。 通过移液管用30ml 0.5%(w/v)NP-40的PBS重悬沉淀   在冰上孵育混合物5分钟
    5. 在4℃下以1,000xg离心10分钟。 取出上清液。
    6. 重悬沉淀(含细胞核)在30毫升PBS,并通过裂解   三个循环的冷冻(干冰或-80℃,10分钟),解冻(37℃ 水浴,3-4分钟)和1分钟涡旋
    7. 将裂解物保存在冰上。 使溶胞产物通过23号皮下注射针至少20次。
    8. 向裂解物中加入10%(w/v)NP-40的PBS溶液,使最终浓度为2%NP-40。 在4℃孵育过夜。
    9. 在4℃下以1500×g离心5分钟以除去大的碎片。
    10. 取在SW28管中的5ml 25%蔗糖(w/v在PBS中),然后注射5ml 50%蔗糖(w/v,在PBS中),用注射器到达试管的底部 制成双层蔗糖垫。 轻轻加载裂解液(上清液   含有衣壳)在垫上用移液管和离心机 80,000 x g 1小时。
    11. 在两者的接口处收集频带 蔗糖层。 用PBS稀释两到三次,加载在a 连续15%至50%(w/v,在PBS中)蔗糖密度梯度。 装载量少 比每个梯度管2ml样品; 使用多管如果有更多   样品。 以80,000×g离心1小时。
    12. 收集乐队 的衣壳,用PBS在SW41管中稀释至管的全部体积, 以80,000×g离心1小时以沉淀衣壳。 对于疱疹病毒 衣壳纯化,有时可以鉴定三条带。 他们 包含顶部,中部和底部带的A-,B-和C-衣壳 分别。  
    13. 倒出上清液,擦去所有液体 管的内壁用Kimwipes TM 薄纸和镊子。 加入10-30微升PBS,在冰上孵育至少2小时或过夜,和 然后用移液器重悬沉淀
    14. 通过负染色电子显微镜或冷冻电子显微镜检查样品的纯度和浓度。

食谱

  1. 疱疹病毒培养基
    含有10%胎牛血清的Dulbecco改良的Eagle培养基

致谢

该协议改编自我们先前发表的论文Dai等人(2013)。 当参考协议时,也鼓励引用本原始文件。 这项工作是由国家卫生研究院(NIH)授予Z.洪周(AI069015)的资助。  

参考文献

  1. Dai,X.,Yu,X.,Gong,H.,Jiang,X.,Abenes,G.,Liu,H.,Shivakoti,S.,Britt,WJ,Zhu,H.,Liu, ,ZH(2013)。 最小的衣壳蛋白介导基本膜蛋白pp150的结合以稳定人巨细胞病毒中含DNA的衣壳 。 PLoS Pathog 9(8):e1003525。
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。

X


How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Dai, X. and Zhou, Z. H. (2014). Purification of Herpesvirus Virions and Capsids. Bio-protocol 4(15): e1193. DOI: 10.21769/BioProtoc.1193; Full Text
  2. Dai, X., Yu, X., Gong, H., Jiang, X., Abenes, G., Liu, H., Shivakoti, S., Britt, W. J., Zhu, H., Liu, F. and Zhou, Z. H. (2013). The smallest capsid protein mediates binding of the essential tegument protein pp150 to stabilize DNA-containing capsids in human cytomegalovirus. PLoS Pathog 9(8): e1003525.




  3. 可重复性反馈:

    • 添加图片
    • 添加视频

    我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

    问题&解答:

    • 添加图片
    • 添加视频

    (提问前,请先登陆)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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


    登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook