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Mar 2020

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Giant Mimiviridae CsCl Purification Protocol
拟菌病毒科巨型病毒的CsCl纯化方案   

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Abstract

While different giant viruses’ purification protocols are available, they are not fully described and they use sucrose gradient that does not reach an equilibrium. Here, we report a protocol for the purification of members of the Mimiviridae family virions resulting from Acanthamoeaba castellanii infections. Viruses are harvested after cell lysis and purified through a high density CsCl gradient to optimize the isolation of the virus from the cell debris or other potential contaminants. Due to the large size of the virion capsids, reaching half a micrometer diameter, the quality of the process can be monitored by light microscopy. The resulting purified particles can then be used to perform new infections, DNA extraction, structural studies, sugar composition analyses, sub-compartment characterization or proteomic experiments.

Keywords: Giant viruses (巨型病毒), Mimivirus (拟菌病毒), CsCl density gradient (氯化铯密度梯度), Virion purification (病毒粒子纯化), Ultracentrifugation (超速离心)

Background

The discovery of Mimivirus, the first virus visible under a light microscope, overlapping in size and genome complexity with unicellular organisms, initiated a new research area in virology (La Scola et al., 2003 ; Raoult et al., 2004). Over the past 15 years, many additional members of the Mimiviridae family have been isolated from various environments and several protocols have been published to purify the virions (La Scola et al., 2003; Byrne et al., 2009; Arslan et al. ; Philippe et al., 2013 ; Campos et al., 2014 ; Andrade et al., 2017). Different approaches were developed, mostly involving sucrose cushion (Campos et al., 2014 ; Andrade et al., 2017), or sucrose discontinuous gradients (Arslan et al., 2011). However, these protocols are not optimal since the density of the virus is higher than the maximum density of a sucrose solution (1.36 g/cm3 and 1.3 g/cm3, respectively – ICTV 9th report, 2011), meaning the equilibrium cannot be reached. Thus, a long time or high speed centrifugation results in a viral pellet instead of a ring or both depending on the conditions used. We recently reported (Jeudy et al., 2019) an optimized version of our previously published protocol using a CsCl discontinuous gradient (Byrne et al., 2009). Here, we provide the detailed protocol for the purification of Mimiviridae particles.

Materials and Reagents

General use

  1. Standard pipette filter tips (TipOne filter tips) (StarLab, catalog numbers: S1120-3810 , S1120-1810 , S1120-8810 and S1112-1720 or equivalent)
  2. Pipettes (10 ml serological pipette) (Sarstedt, catalog number: 86.1254.001 )
  3. Ultrapure water

Cell culture
  1. 175 cm2 flasks (Greiner Bio-One, catalog number: 660175 , or equivalent)
  2. Filtropur S 0.2 (Sarstedt, catalog number: 83.1826.001 )
  3. Stericup Quick Release Express Plus 0.22 µm PES (Merck Millipore, catalog number: S2GPU11RE )
  4. Acanthamoeba castellanii (Douglas) Page (ATCC, catalog number: 30010 )
  5. D-(+)-glucose (Sigma, catalog number: G8270 )
  6. Sodium citrate tribasic trihydrate (Sigma, catalogue number: C7254 )
  7. Proteose-Peptone (Sigma, catalog number: 82450 )
  8. Yeast extract (Fisher BioReagent, catalog number: BP1422 )
  9. Ampicillin Sodium Salt, Cell Culture/Molecular Biology Grade (Euromedex, catalog number: EU0400 )
  10. Kanamycin Sulfate, Cell Culture Grade (Euromedex, catalog number: UK0010 )
  11. D-(+)-Glucose (Sigma, catalog number: G8270 )
  12. Proteose Peptone Yeast Extract medium (PPYG; see Recipes)
    Base medium
    Complete medium
  13. 36% Glucose solution (see Recipes)
  14. Ampicillin stock solution (100 mg/ml) (see Recipes)
  15. Kanamycin stock solution (25 mg/ml) (see Recipes)

Gradient preparation and recovery
  1. Plastic Syringes (5 ml syringe) (Terumo, catalog number: SS*05SE1 )
  2. 21G x 11/2” needle (Terumo, catalog number: AN*2138R1 or equivalent)
  3. Sterile 50 ml conical tubes (Sarstedt, catalog number: 62.547.254 , or equivalent)
  4. Polyallomer Tubes, 38.5 ml (Beckman, catalog number: 326823 )
  5. UltraPure Cesium chloride Optical Grade (Invitrogen, catalog number: 15507-023 )
  6. Cesium chloride solutions (1.2 g/cm3, 1.3 g/cm3, 1.4 g/cm3 and 1.5 g/cm3; see Recipes)
  7. K36 buffer (see Recipes)

Purification quality control
  1. Glass microscope slide (Biosigma, catalog number: VBS653/A )
  2. Coverslip (Biosigma, catalog number: VBS636 )
  3. UVette 220-1600 nm (Eppendorf, catalog number: 952010051 )
  4. Formvar and carbon coated 200 mesh copper/rhodium grids (Electron Microscopy Sciences, catalog number: FCF200-Cu )
  5. Uranyl acetate 1% in water
    Note: Uranyl acetate is a radioactive chemical. It is provided by electron microscopy facilities as we are not allowed to use it in the lab.

Equipment

  1. Microbiological safety hood (ADS laminaire, model: Optimal 12 , or equivalent)

  2. Incubator Bio Performance (Froilabo, catalog number: BP240 , or equivalent)

  3. Centrifuge (Beckman Coulter, model: JXN-30 )

  4. Swinging-Bucket Rotor (Beckman Coulter, model: JS24-38 )

  5. Spectrophotometer (Eppendorf, model: Biophotometer )

  6. Inverted microscope (Zeiss, model: Axio Observer.Z1 )

  7. Transmission Electron Microscope (FEI, model: Tecnai G2 )

  8. Autoclave (SHP Steriltechnik SG, model: Laboklav 135 )

  9. Peristaltic pump (KNF Lab, model: Laboport )

Procedure

  1. Seed 20 175 cm2 flasks with 1,000 Acanthamoeba castellanii cells per cm2 in 20 ml of PPYG medium. Incubate at 32 °C for two days.

    Note: You can add ampicillin and kanamycin at final concentrations of 0.1 mg/ml and 0.025 mg/ml, respectively, in each flask to avoid bacterial contamination.

  2. After two days, the cell confluence should be between 130,000 and 180,000 cells/cm2. Infect the cells with a Mimivirus stock at a multiplicity of infection of 0.2.

    Incubate at 32 °C for two days or until cell lysis is complete.

    Note: There is no need to change the medium before infection or to remove the virus from the flasks.

  3. Harvest the cell debris-containing supernatants from the flasks and pool them into 50 ml conical tubes.

    Note: For 20 175 cm2 flasks containing 20 ml of medium you will need 7 tubes.

  4. Centrifuge at 500 x g for 10 min at room temperature (~25 °C, RT) to remove the cell debris.

  5. Decant the supernatant into new 50 conical tubes.

  6. Centrifuge at 10,000 x g for 25 min at RT to pellet the virus.

  7. Discard the supernatant.

  8. Resuspend the resulting virus pellets in 10 ml K36 buffer by pipeting and pool in two new 50 ml conical tubes (35 ml per tube). Complete the volume to 50 ml with K36 buffer.

  9. Centrifuge at 10,000 x g for 25 min at RT to pellet the virus.

  10. During the centrifugation, prepare four CsCl gradients in Beckman polyallomer tubes. Add 7.5 ml of CsCl solution density 1.5 g/cm3 at the bottom of each tube, then carefully overlay with 9 ml of 1.4 g/cm3 CsCl solution and 9 ml of 1.3 g/cm3 CsCl solution, dropwise. Hold for the final virus-containing 1.2 g/cm3 density layer.

  11. Discard the supernatant from the centrifugation in Step 9.

  12. Resuspend the two viral pellets with 16 ml of 1.2 g/cm3 CsCl solution for each pellet by pipeting.

  13. Overlay 8 ml of resuspended virus pellets on top of each of the four gradients, dropwise (Figure1A).

  14. Centrifuge overnight (between 16 and 18 h) at 100,000 x g at 20 °C in a Beckman Coulter JS24-38 rotor.

  15. After centrifugation, harvest the white ring corresponding to the virus fraction (Figure 1B) by carefully pipetting through the gradient or by puncturing the polyallomer tube and aspirating with a needle and a syringe (Figure 2). Transfer the virus fraction to two new 50 ml conical tubes, add K36 buffer up to 50 ml in each tube and mix by inverting the tubes.



    Figure 1. Centrifuge tubes before (A) and after (B) separation of Mimivirus particles on CsCl gradient. The white ring indicated by the black arrow corresponds to Mimivirus particles.



    Figure 2. Collection of the viral fraction by side puncture


  16. Centrifuge at 10,000 x g for 25 min at RT to pellet the virus.

  17. Resuspend the two pellets with 10 ml of K36 buffer each, complete the volume to 50 ml with K36 and centrifuge them at 10,000 x g for 25 min at RT to wash the virus.

  18. Repeat Steps 16 and 17 twice. For the last wash, pool the resuspended virus in a single 50 ml conical tube.

  19. Resuspend the final pellet in 10 ml of K36 buffer.

  20. The virus is ready to be titrated or stored at -80 °C until further use.


Notes:

  1. Typically, when purifying Acanthamoeba polyphaga Mimivirus using that protocol, we recover 10 ml of viral solution containing 1 x 1010 to 4 x 1010 particles/ml.

  2. The purified particles are infectious and can be used to performed new infections or any other experiments.

Data analysis

  1. Light microscopy

    Drop 4.5 µl of the purified virus suspension on a microscope slide and overlay with a coverslip. Invert for a few hours in order to let the virus settle onto the coverslip for easier focusing. Observe with a light microscope using the 63x objective to confirm the purified virus does not contain any obvious contaminants (Figure 3).



    Figure 3. Observation of Mimivirus purified particles with a light microscope (63x objective, 1.6x optovar)


  2. Transmission Electron Microscopy (TEM)

    For TEM, dilute the purified Mimivirus sample 1:2 (v/v) in K36 buffer and incubate a 10 µl droplet on a formvar- and carbon-coated 200 mesh copper/rhodium grid for 1 min at RT. Wash the grid with three successive 1% uranyl acetate droplets. Leave the residual uranyl acetate on the grid for 1 min and remove by gently touching the edge of the grid with a filter paper. After drying, examine the grid using an electron microscope (Figure 4).



    Figure 4. Observations of Mimivirus purified particles by negative staining

Recipes

  1. Glucose solution

    Dissolve 36 g of glucose and 2 g of sodium citrate in 100 ml of slightly warm water.

    When the solution is clear, use it to prepare the complete PPYG medium.

  2. Proteose Peptone Yeast Extract (PPYG) medium

    Base medium:

    Autoclave 40 g of proteose peptone and 2 g of yeast extract diluted in 1.8 L of ultrapure water in a 2 L glass bottle.

    Let it cool at room temperature.

    Complete medium:

    1. Add the following to the basal medium

      20 ml of MgSO4 400 mM

      16 ml of CaCl2 50 mM

      20 ml of Fe(NH4)2(SO4)2 5 mM

      20 ml of Na2HPO4 250 mM

      20 ml of KH2PO4 250 mM

      and 100 ml of glucose solution.

    2. Filter through 0.22 µm Stericups using a peristaltic pump and store at room temperature

  3. Ampicillin stock solution (100 mg/ml)

    1. Dissolve 1 g of ampicillin in 10 ml of ultrapure water

    2. Filter through a 0.22 µm filter plugged on a syringe

    3. Aliquot in 1.5 ml tubes and store at -20 °C

  4. Kanamycin stock solution (25 mg/ml)

    1. Dissolve 250 mg of kanamycin in 10 ml of ultrapure water

    2. Filter through a 0.22 µm filter plugged on a syringe

    3. Aliquot in 1.5 ml tubes and store at -20 °C

  5. K36 buffer

    1. Dissolve 2.25 g of KH2PO4, 5.8 g of K2HPO4·3H2O, 7.4 g of KCl and 0.9 g of NaCl in 1 L of ultrapure water

    2. Filter through a 1 L 0.22 µm Stericup linked to a peristaltic pump and store at room temperature

  6. Cesium chloride solutions

    Density 1.5 g/cm3: dissolve 22.7 g of CsCl in 27.3 ml of ultra pure water

    Density 1.4 g/cm3: dissolve 19.4 g of CsCl in 30.6 ml of ultra pure water

    Density 1.3 g/cm3: dissolve 15.62 g of CsCl in 34.38 ml of ultra pure water

    Density 1.2 g/cm3: dissolve 11.2 g of CsCl in 38.8 ml of ultra pure water

    Once the solutions appear clear, filter through a 0.22 µm filter plugged on a syringe

Acknowledgments

This work was supported by the French National Center for Scientic Research and the “coup de chapeau” of the Mediterranneen Institute of Microbiology.

Competing interests

There are no conflicts of interest or competing interest.

References

  1. Andrade, A., Rodrigues, R. A. L., Oliveira, G. P., Andrade, K. R., Bonjardim, C. A., La Scola, B., Kroon, E. G. and Abrahão, J. S. (2017). Filling knowledge gaps for mimivirus entry, uncoating, and morphogenesis. J Virol 91(22).
  2. Arslan, D., Legendre, M., Seltzer, V., Abergel, C. and Claverie, J. M. (2011). Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. Proc Natl Acad Sci U S A 108(42): 17486-17491.
  3. Byrne, D., Grzela, R., Lartigue, A., Audic, S., Chenivesse, S., Encinas, S., Claverie, J. M. and Abergel, C. (2009). The polyadenylation site of Mimivirus transcripts obeys a stringent'hairpin rule. Genome Res 19(7): 1233-1242.
  4. Campos, R. K., Boratto, P. V., Assis, F. L., Aguiar, E. R., Silva, L. C., Albarnaz, J. D., Dornas, F. P., Trindade, G. S., Ferreira, P. P., Marques, J. T., Robert, C., Raoult, D., Kroon, E. G., La Scola, B. and Abrahão, J. S. (2014). Samba virus: a novel mimivirus from a giant rain forest, the Brazilian Amazon. Virol J 11: 95.
  5. ICTV 9th report. (2011). https://talk.ictvonline.org/ictv-reports/ictv_9th_report/dsdna-viruses-2011/w/dsdna_viruses/117/mimiviridae.
  6. Jeudy, S., Bertaux, L., Alempic, J. M., Lartigue, A., Legendre, M., Belmudes, L., Santini, S., Philippe, N., Beucher, L., Biondi, E. G., Juul, S., Turner, D. J., Couté, Y., Claverie, J. M. and Abergel, C. (2020). Exploration of the propagation of transpovirons within Mimiviridae reveals a unique example of commensalism in the viral world. ISME J 14(3): 727-739.
  7. La Scola, B., Audic, S., Robert, C., Jungang, L., de Lamballerie, X., Drancourt, M., Birtles, R., Claverie, J. M. and Raoult, D. (2003). A giant virus in amoebae. Science 299(5615): 2033.
  8. Philippe, N., Legendre, M., Doutre, G., Coute, Y., Poirot, O., Lescot, M., Arslan, D., Seltzer, V., Bertaux, L., Bruley, C., Garin, J., Claverie, J. M. and Abergel, C. (2013). Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science 341(6143): 281-286.
  9. Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M. and Claverie, J. M. (2004). The 1.2-megabase genome sequence of Mimivirus. Science 306(5700): 1344-1350.

简介

[摘要]虽然有不同的巨型病毒纯化方案,但它们并没有被完全描述,它们使用的蔗糖梯度不能达到平衡。在这里,我们报告了一个方案,用于纯化由棘球绦虫感染引起的拟病毒科成员病毒。病毒在细胞裂解后收获,并通过高密度CsCl梯度进行纯化,以优化从细胞碎片或其他潜在污染物中分离病毒。由于病毒粒子衣壳尺寸大,直径可达半微米,因此可以通过光学显微镜来监控工艺的质量。由此得到的纯化颗粒可用于进行新的感染、DNA提取、结构研究、糖成分分析、亚组分鉴定或蛋白质组学实验。

[背景]Mimivirus是第一种在光学显微镜下可见的病毒,在大小和基因组复杂度上与单细胞生物重叠,这一发现开创了病毒学的新研究领域(La Scola et al.,2003;Raoult et al.,2004)。在过去的15年里,许多额外的米病毒科成员被从各种环境中分离出来,并且已经发布了几种纯化病毒离子的方案(La Scola等人,2003年;Byrne等人,2009年;Arslan等人,2011年;Philippe等人,2013年;Campos等人,2014年;Andrade等人,2017年)。开发了不同的方法,主要涉及蔗糖缓冲(Campos et al.,2014;Andrade et al.,2017)或蔗糖不连续梯度(Arslan et al.,2011)。然而,由于病毒的密度高于蔗糖溶液的最大密度(分别为1.36 g/cm3和1.3 g/cm3–ICTV第9次报告,2011年),因此这些方案并非最佳方案,这意味着无法达到平衡。因此,长时间或高速离心产生病毒颗粒而不是环或两者都取决于使用的条件。我们最近报告了(Jeudy等人,2019年)我们先前发布的使用CsCl不连续梯度的协议的优化版本(Byrne等人,2009年)。在这里,我们提供了详细的方案纯化咪咪病毒科粒子。

关键字:巨型病毒, 拟菌病毒, 氯化铯密度梯度, 病毒粒子纯化, 超速离心

材料和试剂
 
一般用途
1.    标准移液管过滤器头(TipOne过滤器头)(StarLab,目录号:S1120-3810、S11201810、S1120-8810和S1112-1720或同等产品)
2.    移液管(10毫升血清移液管)(Sarstedt,目录号:86.1254.001)3。超纯水
 
细胞培养
1.    175 cm2烧瓶(Greiner Bio One,目录号:660175,或同等产品)
2.    Filtropur S 0.2(Sarstedt,目录号:83.1826.001)
3.    Stericup快速释放快速加0.22µm PES(默克Millipore,目录号:S2GPU11RE)
4.    卡斯特拉尼棘阿米巴(道格拉斯)页码(ATCC,目录号:30010)
5.    D-(+)-葡萄糖(西格玛,目录号:G8270)
6.    三水合柠檬酸钠(Sigma,产品编号:C7254)
7.    蛋白胨(Sigma,目录号:82450)
8.    酵母抽提物(Fisher生物反应器,目录号:BP1422)
9.    氨苄西林钠盐,细胞培养/分子生物学等级(Euromedex,目录号:EU0400)
10.  硫酸卡那霉素,细胞培养级(Euromedex,目录号:UK0010)
11.  D-(+)-葡萄糖(西格玛,目录号:G8270)
12.  蛋白胨酵母抽提物培养基(PPYG;见配方)
基础培养基
完全培养基
13.  36%葡萄糖溶液(见配方)
14.  氨苄西林储备液(100 mg/ml)(见配方)
15.  卡那霉素原液(25 mg/ml)(见配方)
 
梯度制备与回收
1.     塑料注射器(5毫升注射器)(Terumo,目录号:SS*05SE1)
2.     21G x 11/2“针头(Terumo,目录号:AN*2138R1或同等产品)
3.     无菌50毫升锥形管(Sarstedt,目录号:62.547.254,或同等产品)
4.     高分子管,38.5 ml(Beckman,目录号:326823)
5.     光学级超纯氯化铯(Invitrogen,目录号:15507-023)
6.     氯化铯溶液(1.2 g/cm3、1.3 g/cm3、1.4 g/cm3和1.5 g/cm3;见配方)
7.     K36缓冲液(见配方)
        
净化质量控制
1.     玻璃显微镜载玻片(Biosigma,目录号:VBS653/A)
2.     封面纸(Biosigma,目录号:VBS636)
3.     UVette 220-1600 nm(Eppendorf,目录号:952010051)
4.     Formvar和碳涂层200目铜/铑网格(电子显微镜科学,目录号:FCF200 Cu)
5.     水中乙酸铀酰1%
注:醋酸铀酰是一种放射性化学物质。它是由电子显微镜设备提供的,因为我们不允许在实验室使用它。
 
设备
 
1.    微生物安全罩(ADS laminaire,型号:Optimal 12,或同等产品)
2.    培养箱生物性能(Froilabo,目录号:BP240,或同等产品)
3.    离心机(Beckman Coulter,型号:JXN-30)
4.    摆斗转子(Beckman Coulter,型号:JS24-38)
5.    分光光度计(Eppendorf,型号:生物光度计)
6.    倒置显微镜(蔡司,型号:Axio Observer.Z1)
7.    透射电镜(FEI,型号:Tecnai G2)
8.    高压灭菌器(SHP Steriltechnik SG,型号:Laboklav 135)
9.    蠕动泵(KNF Lab,型号:Laboport)
 
程序
 
1.    在20毫升PPYG培养基中播种20个175平方厘米的烧瓶,每平方厘米有1000个卡斯特拉尼棘阿米巴细胞。在32°C下培养两天。
注:您可以在每个烧瓶中分别添加最终浓度为0.1mg/ml和0.025mg/ml的氨苄西林和卡那霉素,以避免细菌污染。
2.    两天后,细胞汇合应在130000到180000个细胞/cm2之间。以0.2倍的感染倍数用模拟病毒库感染细胞。
在32°C下培养两天或直到细胞完全溶解。
注意:感染前无需更换培养基,也无需从烧瓶中取出病毒。
3.    从烧瓶中收集含有上清液的细胞碎片,并将其汇集到50毫升锥形管中。
注:对于含有20ml培养基的20个175 cm2烧瓶,您需要7个试管。
4.    在室温(~25°C,RT)下,在500 x g下离心10分钟,以去除电池碎片。
5.    将上清液倒入新的50个锥形管中。
6.    在室温下以10000 x g离心25分钟以使病毒颗粒化。
7.    丢弃上清液。
8.    通过移液管将产生的病毒颗粒重新悬浮在10毫升K36缓冲液中,并在两个新的50毫升锥形管(每管35毫升)中汇集。用K36缓冲液定容至50毫升。
9.    在室温下以10000 x g离心25分钟以使病毒颗粒化。
10.  在离心过程中,在Beckman聚丙烯管中制备四个CsCl梯度。在每根管底部添加7.5 ml密度为1.5 g/cm3的CsCl溶液,然后逐滴小心地覆盖9 ml 1.4 g/cm3 CsCl溶液和9 ml 1.3 g/cm3 CsCl溶液。等待最终病毒含有1.2g/cm3的密度层。
11.  丢弃步骤9中离心分离的上清液。
12.  用移液管将两个病毒颗粒用16 ml 1.2 g/cm3 CsCl溶液重新投加。
13.  将8毫升再悬浮病毒颗粒逐滴覆盖在四个梯度的顶部(图1A)。
14.  在Beckman Coulter JS2438转子中,在10万x g、20°C下离心过夜(16至18小时)。
15.  离心后,通过梯度小心地用吸管或刺穿多聚物管并用针头和注射器抽吸(图2),收获与病毒部分(图1B)相对应的白色环。将病毒部分转移到两个新的50毫升锥形管中,在每个管中添加高达50毫升的K36缓冲液,并通过倒置试管进行混合。
 
  
图1。离心管分离前(A)后(B)模拟病毒颗粒在CsCl梯度上分离。黑色箭头所示的白色环对应于拟态病毒粒子。
 
  
图2。侧刺法收集病毒组分
 
16.  在室温下以10000 x g离心25分钟以使病毒颗粒化。
17.  用10ml的K36缓冲液将两个小球再悬浮,用K36使其体积达到50ml,并在室温下以10000 x g离心25分钟以洗涤病毒。
18.  重复步骤16和17两次。最后一次清洗时,将再悬浮的病毒集中在一个50毫升的锥形管中。
19.  在10ml K36缓冲液中再悬浮最终颗粒。
20.  病毒可在-80°C下滴定或储存,直到进一步使用
 
笔记: 
a。通常,当使用该方案纯化棘阿米巴多噬拟态病毒时,我们回收10毫升含有1×1010至4×1010粒子/毫升的病毒溶液。
b。纯化后的颗粒具有传染性,可用于进行新的感染或任何其他实验。
 
数据分析
 
1光学显微镜
将4.5µl纯化病毒悬浮液滴在显微镜载玻片上,并覆盖一张盖玻片。倒置几个小时,让病毒沉淀在盖玻片上,以便更容易聚焦。用63x物镜用光学显微镜观察,确认纯化后的病毒不含任何明显的污染物(图3)。
 
  
图3。用光学显微镜观察拟态病毒纯化颗粒(63x物镜,1.6x视差)
 
2透射电子显微镜
对于透射电镜,在K36缓冲液中稀释纯化的咪咪病毒样品1:2(v/v),并在室温下将10µl液滴培养在formvar和碳涂层的200目铜/铑网格上1分钟。用三个连续的1%乙酸铀酰液滴清洗网格。将残留的醋酸铀酰留在网格上1分钟,然后用滤纸轻轻触摸网格边缘,将其清除。干燥后,用电子显微镜检查网格(图4)。
 
  
图4。咪咪病毒纯化颗粒的阴性染色观察
 
食谱
 
1.    葡萄糖溶液
将36克葡萄糖和2克柠檬酸钠溶于100毫升微温水中。
溶液澄清后,用其配制完整的PPYG培养基。
2.    蛋白胨酵母提取物(PPYG)培养基基:
高压灭菌40 g蛋白胨和2 g酵母抽提物,稀释于1.8 L超纯水中,置于2 L玻璃瓶中。
让它在室温下冷却。
完整介质:
a、 在基础培养基中加入以下物质
20ml硫酸镁400mm
16毫升50毫米氯化钙
20毫升铁(NH4)2(SO4)2 5毫米
20ml Na2HPO4 250mm 20ml KH2PO4 250mm和100ml葡萄糖溶液。
b、 使用蠕动泵过滤0.22µm灭菌杯,并在室温下储存
3.    氨苄西林储备液(100 mg/ml)
a、 将1g氨苄西林溶于10ml超纯水中
b、 通过插在注射器上的0.22µm过滤器过滤
c、 在1.5 ml试管中等分,并在-20°c下储存
4.    卡那霉素原液(25 mg/ml)
a、 将250毫克卡那霉素溶于10毫升超纯水中
b、 通过过滤器22μm的注射器堵塞
c、 在1.5 ml试管中等分,并在-20°c下储存
5.    K36缓冲器
a、 在1L超纯水中溶解2.25g KH2PO4、5.8g K2HPO4·3H2O、7.4g KCl和0.9g NaCl
b、 通过与蠕动泵相连的1L 0.22µm灭菌杯过滤,并在室温下储存
6.    氯化铯溶液
密度1.5 g/cm3:在27.3 ml超纯水中溶解22.7 g CsCl
密度1.4 g/cm3:在30.6 ml超纯水中溶解19.4 g CsCl
密度1.3g/cm3:将15.62g CsCl溶解于34.38ml超纯水中
密度1.2g/cm3:在38.8ml超纯水中溶解11.2g CsCl
溶液澄清后,通过插在注射器上的0.22µm过滤器进行过滤
 
致谢
 
这项工作得到了法国国家科学研究中心和地中海微生物研究所的“起首政变”的支持。
        
相互竞争的利益相互竞争的利益
 
不存在利益冲突或利益冲突。
 
工具书类
 
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引用:Bertaux, L., Lartigue, A. and Jeudy, S. (2020). Giant Mimiviridae CsCl Purification Protocol. Bio-protocol 10(22): e3827. DOI: 10.21769/BioProtoc.3827.
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