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Enterovirus 71 Virus Propagation and Purification
肠道病毒71的增殖和纯化   

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Abstract

Since its discovery in 1969, enterovirus 71 (EV71) has emerged as a serious worldwide health threat. This member of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. This is the propagation and purification procedure to produce infectious virion.

Keywords: Virus purification(病毒纯化), Gradient(梯度), Picornavirus(小核糖核酸病毒), Procapsid(原壳体)

Materials and Reagents

  1. Hela cells
  2. EV71 inoculum (ATCC, www.atcc.org)
  3. DMEM (Sigma-Aldrich, catalog number: SH3002201 )
  4. Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: SH3039603 )
  5. Polyethylene glycol (PEG) 8000 (Thermo Fisher Scientific, catalog number: P156-3 )
  6. Tris-HCl
  7. Magnesium chloride (MgCl2)
  8. 0.5 M NaCl
  9. 0.05 mg/ml DNase (Thermo Fisher Scientific, catalog number: NC9709009 )
  10. 0.1 M EDTA (pH=8.0)
  11. Ammonium hydroxide (Sigma-Aldrich, catalog number: A5132-5 Kg )
  12. Potassium tartrate (Sigma-Aldrich, catalog number: 25516-500 g )
  13. 100 kD cutoff spin column with 4 ml capacity (Millipore, catalog number: UFC810096 )
  14. 30% sucrose-cushion in purification buffer
  15. Purification buffer (see Recipes)

Equipment

  1. Cell Stacks (1 10-stack and 1 2-stack) (Thermo Fisher Scientific, catalog numbers: 12-567-303 and 12-567-301 )
  2. Centrifuge
  3. SLA1500 rotor
  4. 50.2ti rotor
  5. 26.3 ml red-capped Beckman tubes (Beckman Coulter, catalog number: 355618 )
  6. SW41 rotor
  7. Beckman Ultra-Clear centrifuge tubes (14 x 89 mm) (Beckman Coulter, catalog number: 344059 )
  8. Chemistry ring stand with clamp
  9. Small light source
  10. Microcentrifuge

Procedure

  1. 90-95% confluent Hela cell monolayers (passage number not exceeding 30) grown in cell stacks (1 10-stack and 1 2-stack) were infected with EV71 strain 1095/Shiga at an MOI of 0.1 in culture with 600 ml of DMEM (500 ml in 10-stack and 100 ml in 2-stack). Virus was allowed to attach and infect for one hour at 37 °C. 600 ml of DMEM supplemented with 5% fetal bovine serum (500 ml in 10-stack and 100 ml in 2-stack) was added to achieve a final volume of 1,200 ml total (1,000 ml in 10-stack and 200 ml in 2-stack). Infections were allowed to continue at 37 °C.
  2. Cells and media were harvested 24 h post infection, (or as soon as cytopathic effects were obvious) and subjected to three freeze-thaw cycles. To remove cell debris, the lysate was centrifuged at 13,000 rpm in a SLA1500 rotor at 4 °C for 15 min. To the lysate, PEG 8000 was added to provide a final concentration of 8% and NaCl, was added to provide a final concentration of 0.5 M. Virus was precipitated overnight at 4 °C with stirring and then centrifuged in a SLA1500 rotor (4 °C, 13,000 rpm, 45 min).
  3. Pellets were resuspended in 10 ml purification buffer, bottles were rinsed with 5 ml purification buffer, and 0.05 mg/ml DNase was added (do not exceed total buffer volume of 20 ml). The suspension was incubated at room temperature for 10 min with gentle rocking.
  4. After incubation, 0.1 M EDTA (pH=8.0) was added (10% total volume), the pH was adjusted using ammonium hydroxide, and supernatant was cleared by low speed centrifugation (4,000 rpm, 5 min, 4 °C).
  5. The supernatant was transferred to a red-capped Beckman tube. 2 ml of 30% sucrose in purification buffer was pipetted directly underneath the virus-containing supernatant. The virus was then pelleted through the sucrose cushion (50.2ti rotor, 48,000 rpm, 4 °C, 2 h).
  6. The pellet was resuspended in 2 ml of purification buffer, tube rinsed with an additional 1 ml of purification buffer, centrifuged at 4,000 rpm for 5 min to remove any remaining cellular debris, and supernatant applied to a 10-35% potassium tartrate (K-Tartrate) step gradient in the Ultra-clear centrifuge tubes for final purification by ultracentrifugation (36,000 rpm, 4 °C, 2 h, SW41 rotor).
  7. The centrifuge tube was immobilized in a clamp on a ring stand above a small light source (Figure 1). Other light sources were extinguished to visualize the two distinct bands of virus, which were collected by side puncture and diluted in purification buffer to reach a final volume of 4 ml. The 4 ml sample was placed into a 100 kD cutoff spin column and the volume was reduced to about 50-100 microliters by centrifugation at 4,000 rpm. The sample was diluted again with purification buffer to return the volume to 4 ml and then centrifuged to reduce volume to 50-100 microliters. This process of washing the sample to remove tartrate and replace it with purification buffer was repeated in three more wash steps (four in total).


    Figure 1. A) Two bands of virus in the tartrate gradient are visible when a small light source is placed beneath the centrifuge tube. B) The upper band contains empty capsids, which have intact VP0 whereas the lower band C) contains native infectious virus. As assayed by negative stain TEM (shown) and PAGE gel (data not shown).

  8. The concentration of the final 100 microliters of virus was determined by spectrometry reading (procapsid read at wavelength of 280 nm and native virus read at a wavelength of 260 nm).
  9. As previously reported the upper band consisted of procapsid, characterized by the presence of uncleaved VP0 and lack of genomic material. The lower band consisted of native virus comprised of VP2 and VP4, with packaged genome. Each species was diluted to a concentration of 0.1 mg/ml for storage. Procapsid is stored at 4 °C and native virus is stored at -80 °C.

Recipes

  1. Purification buffer
    10 mM Tris-HCl
    200 mM NaCl
    50 mM MgCl2
    pH=7.5 (if initial pH is < 7.5, adjust with 1 M NaOH, if initial pH is > 7.5, adjust with 1 M HCl)

Acknowledgments

This work was funded in part by the Pennsylvania Department of Health Tobacco CURE Funds and the Junior Faculty Research Scholar Max Lang Award from the Pennsylvania State University College of Medicine. This protocol was previously described in Shingler et al. (2013).

References

  1. Cifuente, J. O., Lee, H., Yoder, J. D., Shingler, K. L., Carnegie, M. S., Yoder, J. L., Ashley, R. E., Makhov, A. M., Conway, J. F. and Hafenstein, S. (2013). Structures of the procapsid and mature virion of enterovirus 71 strain 1095. J Virol 87(13): 7637-7645.
  2. Shingler, K. L., Yoder, J. L., Carnegie, M. S., Ashley, R. E., Makhov, A. M., Conway, J. F. and Hafenstein, S. (2013). The enterovirus 71 A-particle forms a gateway to allow genome release: a cryoEM study of picornavirus uncoating. PLoS Pathog 9(3): e1003240.

简介

自从1969年发现以来,肠道病毒71(EV71)已经成为严重的全球健康威胁。 小核糖核酸病毒家族的这个成员引起手足口病,并且还具有侵入中枢神经系统以引起严重疾病和死亡的能力。 这是产生感染性病毒粒子的繁殖和纯化过程

关键字:病毒纯化, 梯度, 小核糖核酸病毒, 原壳体

材料和试剂

  1. 赫拉细胞
  2. EV71接种物(ATCC, www.atcc.org
  3. DMEM(Sigma-Aldrich,目录号:SH3002201)
  4. 胎牛血清(Thermo Fisher Scientific,目录号:SH3039603)
  5. 聚乙二醇(PEG)8000(Thermo Fisher Scientific,目录号:P156-3)
  6. Tris-HCl
  7. 氯化镁(MgCl 2)
  8. 0.5 M NaCl
  9. 0.05mg/ml DNA酶(Thermo Fisher Scientific,目录号:NC9709009)
  10. 0.1M EDTA(pH = 8.0)
  11. 氢氧化铵(Sigma-Aldrich,目录号:A5132-5Kg)
  12. 酒石酸钾(Sigma-Aldrich,目录号:25516-500g)
  13. 100kD截留旋转柱,具有4ml容量(Millipore,目录号:UFC810096)
  14. 30%蔗糖垫在净化缓冲液中
  15. 净化缓冲液(参见配方)

设备

  1. 电池堆(110堆和12堆)(Thermo Fisher Scientific,目录号:12-567-303和12-567-301)
  2. 离心机
  3. SLA1500转子
  4. 50.2ti转子
  5. 26.3ml红盖的Beckman管(Beckman Coulter,目录号:355618)
  6. SW41转子
  7. Beckman Ultra-Clear离心管(14×89mm)(Beckman Coulter,目录号:344059)
  8. 与夹子的化学圆环立场
  9. 小光源
  10. 微量离心机

程序

  1. 在培养物中用EV71菌株1095/Shiga以0.1的MOI感染在细胞堆叠(110堆叠和12堆叠)中生长的90-95%汇合的Hela细胞单层(传代数不超过30),用600ml DMEM (10-叠层中500ml,2-叠层中100ml)。 使病毒附着并在37℃感染1小时。 加入600ml补充有5%胎牛血清(500-在10-叠层中和100-在2-叠层中)的DMEM,以获得总共1,200ml的最终体积(1000-叠层中的1,000ml,2- 堆栈)。 在37℃下继续感染。
  2. 感染后24小时收获细胞和培养基(或者一旦细胞病变效应明显),并进行三个冻融循环。为了除去细胞碎片,将裂解物在SLA1500转子中在4℃下以13,000rpm离心15分钟。向裂解物中加入PEG 8000以提供8%的终浓度,并加入NaCl以提供0.5M的最终浓度。病毒在4℃下搅拌下沉淀过夜,然后在SLA1500转子(4° 13,000rpm,45分钟)。
  3. 将沉淀重悬于10ml纯化缓冲液中,用5ml纯化缓冲液冲洗瓶子,并加入0.05mg/ml DNA酶(不超过20ml的总缓冲液体积)。将悬浮液在室温下温和摇动孵育10分钟。
  4. 孵育后,加入0.1M EDTA(pH = 8.0)(10%总体积),使用氢氧化铵调节pH,并通过低速离心(4,000rpm,5分钟,4℃)清除上清液。
  5. 将上清液转移到红盖的Beckman管中。将2ml 30%蔗糖的纯化缓冲液直接吸移到含有病毒的上清液下面。然后将病毒通过蔗糖垫(50.2转子,48,000rpm,4℃,2小时)沉淀。
  6. 将沉淀重悬于2ml纯化缓冲液中,用另外1ml纯化缓冲液冲洗管,以4000rpm离心5分钟以除去任何残留的细胞碎片,将上清液施加于10-35%酒石酸钾(K- (36,000rpm,4℃,2小时,SW41转子)进行最终纯化,在超清离心管中进行梯度洗脱。
  7. 将离心管固定在小型光源上方的环架上的夹具中(图1)。熄灭其它光源以显现病毒的两个不同条带,其通过侧穿刺收集并在纯化缓冲液中稀释以达到4ml的最终体积。将4ml样品置于100kD截留旋转柱中,并通过在4,000rpm离心将体积减少至约50-100微升。再次用纯化缓冲液稀释样品以使体积返回至4ml,然后离心以将体积减少至50-100微升。在另外三个洗涤步骤(总共四个)中重复洗涤样品以除去酒石酸盐并用纯化缓冲液置换的过程。


    图1. A)当将小光源放置在离心管下方时,酒石酸盐梯度中的两条病毒带是可见的。 B)上带含有空衣壳,其具有完整的VP0,而较低的带C)含有天然感染性病毒。通过阴性染色TEM(显示)和PAGE凝胶(数据未显示)测定
  8. 通过光谱读数(在280nm波长下读取的前衣片和在260nm波长下读取的天然病毒)测定最终100微升病毒的浓度。
  9. 如先前报道的,上带由前衣壳组成,其特征在于存在未切割的VP0和缺乏基因组材料。较低的条带由包含VP2和VP4的天然病毒和包装的基因组组成。每种物质稀释至0.1mg/ml的浓度储存。原虫病在4℃储存,天然病毒储存在-80℃。

食谱

  1. 纯化缓冲液
    10mM Tris-HCl
    200 mM NaCl
    50mM MgCl 2/v/v pH = 7.5(如果初始pH <7.5,用1M NaOH调节,如果初始pH> 7.5,用1M HCl调节)。

致谢

这项工作部分由宾夕法尼亚州卫生烟草治疗基金和宾夕法尼亚州立大学医学院的初级学院研究学者Max Lang奖资助。 该协议之前在Shingler等人(2013)中描述。

参考文献

  1. Cifuente,J.O.,Lee,H.,Yoder,J.D.,Shingler,K.L.,Carnegie, S.,Yoder,J.L.,Ashley,R.E.,Makhov,A.M.,Conway,J.F Hafenstein,S.(2013)。 肠衣病毒71菌株1095的前衣壳和成熟病毒粒子的结构。 Virol 87(13):7637-7645。
  2. Shingler,K.L.,Yoder,J.L.,Carnegie,M.S.,Ashley,R.E.,Makhov,A.M.,Conway,J.F.and Hafenstein,S。(2013)。 肠道病毒71颗粒形成了允许基因组释放的途径:小核糖核酸病毒脱壳的cryoEM研究。 PLoS Pathog 9(3):e1003240。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shingler, K. L., Organtini, L. J. and Hafenstein, S. (2014). Enterovirus 71 Virus Propagation and Purification. Bio-protocol 4(9): e1117. DOI: 10.21769/BioProtoc.1117.
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