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An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes
利用PEI优化生产,滴定和中和SARS-CoV spike萤光素酶假病毒的方法   

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Abstract

The protocol outlined represents a cost-effective, rapid and reliable method for the generation of high-titre viral pseudotype particles with the wild-type SARS-CoV spike protein on a lentiviral vector core using the widely available transfection reagent PEI. This protocol is optimized for transfection in 6-well plates; however it can be readily scaled to different production volumes according to application. This protocol has multiple benefits including the use of readily available reagents, consistent, high pseudotype virus production Relative Luminescence Units (RLU) titres and rapid generation of novel coronavirus pseudotypes for research into strain variation, tropism and immunogenicity/sero-prevalence.

Keywords: SARS coronavirus(SARS冠状病毒), Lentiviral pseudotype(慢病毒假型), Virus neutralization(病毒中和)

Background

The production and use of pseudotyped viral particles (PV) is widely established for many viruses, and applications in the fields of serology, surveillance and vaccine development are manifold (Temperton et al., 2015; Carnell et al., 2015). PVs have proven to be powerful tools to study the effects of viral envelope glycoprotein mutations on serological outcomes, viral tropism and immunogenicity studies especially when combined with epitope information. PVs are chimeric viral constructs in which the outer (surface) glycoprotein(s) of one virus is combined with the replication-defective viral ‘core’ of another virus. PV allow for accurate, sequence-directed, sensitive antibody neutralization assays and antiviral screening to be conducted within a low biosecurity facility and offer a safe and efficient alternative to wildtype virus use, making them exquisitely beneficial for many emerging RNA viruses of pandemic potential. Many of the published protocols require modification of the SARS spike glycoprotein and/or expensive transfection reagents (Temperton, 2009). The protocol presented here utilizes the full-length, non-codon-optimized spike protein in conjunction with the low-cost transfection reagent PEI, making this protocol widely applicable to many stakeholder laboratories. Figure 1 shows a cartoon of the lentiviral SARS-CoV PV production process directed by plasmid co-transfection.


Figure 1. Cartoon representation of the production of SARS pseudotypes. HEK293T/17 cells are transfected with three plasmids, bearing the relevant genes (Lentiviral vector, packaging construct and SARS-CoV spike expression plasmid) for the production of SARS-CoV Spike bearing lentiviral pseudotypes. This figure is modified from Carnell et al. (2015).

Materials and Reagents

  1. MultiGuard Barrier pipette tips 1-20 and 1-200 μl (Sorenson BioScience, catalog number: 30550T )
  2. NuncTM Cell-Culture Treated Multidishes (6-well) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  3. Microcentrifuge tube Safe-Lock write-on graduated with lid latch 1.5 ml
  4. Sterile syringes (10 ml), Generic
  5. Millex-HA 0.45 µm filters (Merck, catalog number: SLHAM33SS )
  6. 96-well white plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 136101 )
  7. HEK 293T/17 cells (ATCC, catalog number: CRL-11268 )
  8. Huh7 cells (Cell Signaling Technology, catalog number: 300156 )
  9. Plasmids
    1. Glycoprotein expression plasmid: pCAGGS-SARS-CoV spike
    2. Lentiviral vector expressing firefly luciferase: pCSFLW
    3. Second-generation lentiviral packaging construct plasmid: p8.91 (expresses gag, pol and rev)
    Note: Information on the plasmids above can be found in Temperton et al. (2005) and Carnell et al. (2015). Plasmids available from Viral Pseudotype Unit, University of Kent. n.temperton@kent.ac.uk
  10. Dulbecco’s modified Eagle medium (DMEM) with GlutaMAX (Thermo Fisher Scientific, catalog number: 31966021 ) supplemented with 10% foetal bovine serum (FBS) (Pan-Biotech, catalog number: P40-37500 ) and 1% penicillin/streptomycin (P/S) (Pan Biotech, catalog number: P06-07100 )
  11. Gibco Reduced Serum media Opti-MEM® (Thermo Fisher Scientific, catalog number: 31985047 )
  12. Branched Polyethyleneimine (PEI) solution at concentration of 1 mg/ml (Sigma-Aldrich, catalog number: 408727 ).
    Note: PEI is dissolved in dH2O to a concentration of 1 mg/ml and the pH is adjusted to 7 using diluted (1:3) concentrated HCl.
  13. Phosphate-buffered saline (PBS)
  14. Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  15. Positive control antibody (monoclonal/polyclonal or post-infection serum) that can neutralize the SARS pseudotype
  16. Bright GloTM luciferase assay system (Promega, catalog number: E2650 )

Equipment

  1. Class II biosafety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM )
  2. Water bath or incubator
  3. Pipettes (Gilson, model: PIPETMAN® Classic, P2 , P20 , P200 and P1000 )
  4. Optional: BIO-RAD TC20TM Automated Cell Counter (Bio-Rad Laboratories, catalog number: 1450102EDU )
  5. Plate centrifuge (ELMI, model: SkyLine CM-6MT )
  6. Glomax 96 luminometer (Promega, model: GloMax® 96 )
    Note: This product has been discontinued.

Procedure

  1. Transfection steps
    Note: All steps should be carried out in a class II biosafety cabinet to avoid contamination. Timeline: Transfection–24 h.
    1. 293T/17 cells should be subcultured into 6-well plates at a ratio that will deliver 70-90% confluence at the time of transfection. Typically seeding 4 x 105 cells per well will achieve this level of confluency. Example is shown in Figure 2.


      Figure 2. Example of the confluency expected prior to transfection of HEK293T/17 cells

    Timeline: Day of transfection.
    1. DMEM/10% FBS/1% P/S and Opti-MEM® should be pre-warmed to 37 °C using a water bath or an incubator.
    2. Prepare two labelled sterile 1.5 ml microcentrifuge tubes (tube 1 and tube 2) for each well of a 6-well plate which will be used for transfections.
    3. Add the following plasmid constructs for transfection to tube 1:
      pCAGGS-SARS-CoV spike: 450 ng
      p8.91-lentiviral vector: 500 ng
      pCSFLW: 750 ng
    4. Add 100 µl Opti-MEM® to the plasmid DNA mix (tube 1).
    5. Add 100 µl Opti-MEM® and 17.5 µl of 1 mg/ml PEI to tube 2.
    6. Incubation step: Mix both tubes by gently flicking and incubate for 5 min at room temperature (RT).
    7. After incubation, pipette the Opti-MEM®/PEI solution from tube 2 into the Opti-MEM®/DNA solution in tube 1.
    8. Incubation step: Incubate the tube at RT for 20 min whilst gently flicking the tube to mix every 3-4 min.
    9. Whilst the transfection mix is incubating, the culture media on the 293T/17 cells should be removed and 2 ml of fresh DMEM/10% FBS/1% P/S added per well. It is imperative at this point to add culture media slowly to one side of the well to avoid detaching the adherent cell monolayer.
    10. After 20 min incubation, pipette the DNA/Opti-MEM®/PEI solution onto the 293T/17 cells by adding dropwise throughout the total surface area of the well. Swirl the 6-well plate(s) gently to ensure an even dispersal of reagent mix.
    11. Incubation step: Incubate the plate at 37 °C, 5% CO2 overnight (o/n). In our hands incubation times of between 12-16 h result in equivalent final PV production RLU titres.
    Timeline: 12-16 h post transfection.
    1. Post o/n incubation the culture media on the cells should be carefully removed and 2 ml fresh DMEM/10% FBS/1% P/S added. Again, add media slowly to one side of the well to avoid cell detachment.
    2. Incubate the 6-well plates at 37 °C 5% CO2 o/n for 32-36 h.
    Timeline: 44-52 h post transfection.
    1. Supernatant containing the viral pseudotype particles are harvested using a 2.5 ml sterile syringe and subsequently filtered into Falcon or microcentrifuge tubes via a syringe driven Millex HA-0.45 µm filter.
    2. Store all filtered supernatants at -80 °C until downstream use. It is recommended that supernatant is stored as aliquots to avoid multiple freeze-thaw cycles that may impact viral RLU titres.
      Note: Supernatant may be stored at 4 °C for up to one week with no detectable loss of RLU titre.
    3. Optional step: Additional culture media may be added to cells to allow a second harvest 18-24 h later by adding further DMEM/10% FBS/1% P/S. In this case, extreme care must be taken in initial PV collection (step A15) to avoid damage to the cell monolayer. We have observed that cells in poor health after first harvest result in significantly lower PV production RLU titres upon second harvest.
    Note: A control pseudotype virus can be created by following the steps outlined above but omitting the pCAGGS-SARS-CoV spike construct. This produces particles that do not express a viral surface glycoprotein (Delta Env control, Figure 3).

  1. Titration steps (Figure 3)
    Note: Titration consists of transduction of reporter (in this case firefly luciferase) into target cells mediated by the viral glycoprotein expressed on the viral pseudotype (SARS-CoV spike). Controls for titration are provided via the inclusion of ‘cell only’ and ‘Delta Env’ columns. Control for transduction can be provided via a PV bearing the Vesicular stomatitis virus G protein (VSV-G) which utilizes a ubiquitous receptor which results in high RLU titres in all cell lines tested.


    Figure 3. 96-well plate set-up for pseudotype titration. Serial dilution step showing addition of 100 µl of pseudotype virus supernatant to each well of row A and dilution of 50 µl taken from this well to row B. This process is continued to end of plate (row H) at which point the final 50 µl is discarded. Delta Env control is indicated in red (column 11) and cell only control is indicated in green (column 12). One set of pipette tips can be used per dilution series (plate).

    1. In a 96-well white plate add 50 µl of DMEM/10% FBS/1% P/S to the entire column of ‘cell only’ control (see Figure 3 column 12).
    2. Add 50 µl of DMEM/10% FBS/1% P/S from row B to H that are to contain PV or Delta-Env control virus.
    3. Add 100 µl of SARS pseudotype virus supernatant to each well of row A (excluding control columns) and add 100 µl of Delta-Env to column 11 (see Figure 3).
    4. Remove 50 µl from row A virus-containing wells and perform two-fold serial dilutions down all the wells beneath it.
    5. With each dilution step use a pipette to mix 8 times by pipetting up and down and taking care not to produce air bubbles.
    6. After completing serial dilution the final 50 µl from the final well of each column should be discarded.
      Note: At this point each well should contain 50 µl of mixed DMEM and PV supernatant.
    7. Prepare a plate of susceptible target cells (Huh-7 for SARS PV), preferentially subcultured 1:4 48 h before:
      1. Remove culture media from plate.
      2. Wash the plate twice with 2 ml of PBS and discard.
      3. Add 2 ml of trypsin to the plate to detach cells.
      4. After cells have detached add 6 ml of DMEM/10% FBS/1% P/S to the plate to quench trypsin activity, and resuspend cells gently.
      5. Count cells using TC20TM Automated Cell Counter or haemocytometer and add 1 x 104 cells in a total volume of 50 µl to each well.
    8. Centrifuge plate for 1 min at 800 x g (Rcf) if there are visible droplets on the sides of the wells.
    9. Incubate the plate for 48 h at 37 °C 5% CO2.
    10. Read plate using Bright GloTM luciferase assay system on a Glomax 96 luminometer (or equivalent).

  2. Pseudotype based neutralisation assay (pMN)
    Note: pMN is the Inhibition of PV mediated transduction via an antibody (or inhibitor) directed against the SARS glycoprotein.
    1. In a 96-well white plate add 50 µl of DMEM/10% FBS/1% P/S to rows B to H, columns 1-12.
    2. Add known amount of antibody (example 5 µl HN and R sera in Figure 4) into wells of row A, columns 2-10 in a total volume of 100 µl DMEM/10% FBS/1% P/S. Add known amount (e.g., 5 µl) of positive and negative antisera into wells A11 and A12 as controls.


      Figure 4. Anti-SARS antibodies (post-infection sera HN and R) neutralization of SARS viral pseudotype entry into Huh7 cells. Two repeats are plotted for each serum sample.

    3. Remove 50 µl from row A wells and perform two-fold serial dilutions down all the wells beneath it.
    4. With each dilution step use a pipette to mix 15 times by pipetting up and down and taking care not to produce air bubbles.
    5. After completing serial dilution the final 50 µl from the final well of each column should be discarded.
      Note: At this point each well should contain 50 µl of mixed DMEM and serial dilutions of antibody/inhibitor.
    6. Centrifuge plate for 1 min at 800 x g (Rcf) to ensure no inhibitor or liquid is located on the walls of the well.
    7. Using data obtained from the titration, calculate the amount of DMEM required to dilute your SARS-PV to obtain 1 x 106 RLU in 50 µl, with a total volume of 5 ml. For example with an RLU/ml of 1 x 108, 1 ml of PV should be mixed with 4 ml of DMEM.
    8. Mix this diluted PV solution using the multichannel pipette, and aliquot 50 µl into each well on the plate, with the exception of wells A6-A12 (cell only control). A1-A6 will serve as virus only control.
    9. Centrifuge plate for 1 min at 800 x g (Rcf) to ensure no virus is left on the walls of the well.
    10. Incubate the plates for 1 h at 37 °C 5% CO2, allowing time for the antibody/inhibitor to bind the SARS glycoprotein.
    11. Prepare a plate of susceptible target cells (Huh-7 for SARS PV), preferentially subcultured 1:4 48 h before:
      1. Remove culture media from plate.
      2. Wash the plate twice with 2 ml of PBS and discard.
      3. Add 2 ml of trypsin to the plate to detach cells.
      4. After cells have detached add 6 ml of DMEM/10% FBS/1% P/S to the plate to quench trypsin activity, and resuspend cells gently.
      5. Count cells using TC20TM Automated Cell Counter or haemocytometer and add 1 x 104 cells in a total volume of 50 µl to each well.
    12. Incubate the plate for 48 h at 37 °C 5% CO2.
    13. Read plate using Bright GloTM luciferase assay system on a Glomax 96 luminometer (or equivalent).

Data analysis

  1. RLU readings are multiplied to RLU/ml by the dilution factor of each well (20x, 40x, 80x, 160x, 320x, 640x, 1,280x, 2,560x). The mean of all 8 RLU/ml values is used as the final value reported for that column in the titration step. A linear relationship should be observed between RLU values and PV dilution, with values decreasing by 50% after each 1:2 dilution. Care should be taken to check this linear relationship before multiplication, as this inherently can lead to false positives despite lack of PV or working PV (Table 1).

    Table 1. Analysis of PV titration data. RLU values are multiplied to give an RLU/ml value (example highlighted in green) giving RLU/ml values for each of the dilution points. The mean/average is then calculated from all 8 dilution points. Care must be taken to observe a linear relationship between dilution factor (X factor) and RLU, or multiplication can lead to inflated RLU/ml values (example highlighted in yellow).


  2. Protocol read-out and titration results (Figure 5).


    Figure 5. Results of pseudotype production RLU titres using optimized transfection protocol. SARS; PV with SARS-CoV Spike on surface, Cell; cell only control, Delta Env; PV with no surface glycoprotein, VSV-G; PV with VSV-G on surface.

  3. The protocol outlined provides a rapid and consistent method for the generation of high-titre viral pseudotype particles expressing the SARS-CoV spike protein suitable for further downstream R&D applications. Efficient knock-down (neutralization) of SARS-CoV pseudotype virus entry using two post-infection sera (HN and R) demonstrates potential utility for vaccine immunogenicity and mAb/antiviral screening. The use of readily available reagents should facilitate increased reproducibility.

Acknowledgments

The authors acknowledge Dr Paul Chan (Faculty of Medicine, The Chinese University of Hong Kong) for the two positive post-infection control sera. This work was funded by the Medway School of Pharmacy, Kent, UK. This protocol was modified and improved from PMID: 15757556 and 26587388.

References

  1. Carnell, G. W., Ferrara, F., Grehan, K., Thompson, C. P. and Temperton, N. J. (2015). Pseudotype-based neutralization assays for influenza: a systematic analysis. Front Immunol 6: 161.
  2. Grehan, K., Ferrara, F. and Temperton, N. (2015). An optimised method for the production of MERS-CoV spike expressing viral pseudotypes. MethodsX 13(2): 379-384.
  3. Temperton, N. J. (2009). The use of retroviral pseudotypes for the measurement of antibody responses to SARS coronavirus. In Lal, S. K. (Ed.). Molecular biology of the SARS- coronavirus. Springer.
  4. Temperton, N. J., Chan, P. K., Simmons, G., Zambon, M. C., Tedder, R. S., Takeuchi, Y. and Weiss, R. A. (2005). Longitudinally profiling neutralizing antibody response to SARS coronavirus with pseudotypes. Emerg Infect Dis 11(3): 411-416.
  5. Temperton, N. J., Wright, E. and Scott, S. D. (2015). Retroviral pseudotypes–from scientific tools to clinical utility. eLS 1-11.

简介

概述的方案代表了使用广泛可用的转染试剂PEI在慢病毒载体核心上产生具有野生型SARS-CoV刺突蛋白的高滴度病毒假型颗粒的成本效益,快速和可靠的方法。 该协议针对6孔板中的转染进行了优化; 然而,根据应用,它可以容易地扩展到不同的生产量。 该方案具有多种益处,包括使用容易获得的试剂,一致的,高假型病毒生产相对发光单位(RLU)滴度,并快速产生用于研究菌株变异,趋向性和免疫原性/血清流行性的新型冠状病毒假型。
【背景】假型病毒颗粒(PV)的生产和使用对于许多病毒是广泛建立的,并且在血清学,监测和疫苗开发领域的应用是多方面的(Temperton等人,2015; Carnell等人,2015)。 PV已经被证明是研究病毒包膜糖蛋白突变对血清学结果,病毒向性和免疫原性研究的影响的有力工具,特别是当与表位信息结合时。 PV是嵌合病毒构建体,其中一个病毒的外(表面)糖蛋白与另一病毒的复制缺陷病毒核心组合。 PV允许在低生物安全设施内进行准确的,顺序导向的敏感抗体中和测定和抗病毒筛选,并为野生型病毒使用提供安全有效的替代方案,使其对许多新出现的大流行潜在RNA病毒有益。许多已发表的方案需要修改SARS尖峰糖蛋白和/或昂贵的转染试剂(Temperton,2009)。本文提出的方案利用全长非密码子优化的穗蛋白与低成本转染试剂PEI结合,使该方案广泛适用于许多利益相关者实验室。图1显示了由质粒共转染导致的慢病毒SARS-CoV PV生产过程的漫画。

关键字:SARS冠状病毒, 慢病毒假型, 病毒中和

材料和试剂

  1. MultiGuard屏障移液管提示1-20和1-200μl(Sorenson BioScience,目录号:30550T)
  2. Nunc TM细胞培养处理的多片剂(6孔)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)
  3. 微量离心管安全锁写入刻度与盖子闩1.5毫升
  4. 无菌注射器(10 ml),通用型
  5. Millex-HA 0.45μm过滤器(默克,目录号:SLHAM33SS)
  6. 96孔白板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:136101)
  7. HEK 293T / 17细胞(ATCC,目录号:CRL-11268)
  8. Huh7细胞(Cell Signaling Technology,目录号:300156)
  9. 质粒
    1. 糖蛋白表达质粒:pCAGGS-SARS-CoV spike
    2. 表达萤火虫萤光素酶的慢病毒载体:pCSFLW
    3. 第二代慢病毒包装构建质粒:p8.91(表达gag,pol和rev)
    注意:有关上述质粒的信息可以在Temperton等人(2005)和Carnell等人(2015年)。肯特大学病毒假单位的质粒。 n.temperton@kent.ac.uk
  10. 补充有10%胎牛血清(FBS)(Pan-Biotech,目录号:P40-37500)和1%青霉素/链霉素(P / P)的具有GlutaMAX(Thermo Fisher Scientific,目录号:31966021)的Dulbecco改良Eagle培养基S)(Pan Biotech,目录号:P06-07100)
  11. Gibco减少血清培养基Opti-MEM ®(Thermo Fisher Scientific,目录号:31985047)
  12. 分散聚乙烯亚胺(PEI)溶液,浓度为1mg / ml(Sigma-Aldrich,目录号:408727)。
    注意:将PEI溶解于浓度为1mg / ml的dH (1:3)浓HCl。
  13. 磷酸盐缓冲盐水(PBS)
  14. 胰蛋白酶-EDTA(0.05%),苯酚红(Thermo Fisher Scientific,Gibco ,目录号:25300054)
  15. 可以中和SARS假型的阳性对照抗体(单克隆/多克隆或后感染血清)
  16. Bright Glo TM 荧光素酶测定系统(Promega,目录号:E2650)

设备

  1. II类生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM ,型号:MSC-Advantage TM )
  2. 水浴或孵化器
  3. 移液器(Gilson,型号:PIPETMAN Classic,P2,P20,P200和P1000)
  4. 可选:BIO-RAD TC20 TM 自动细胞计数器(Bio-Rad Laboratories,目录号:1450102EDU)
  5. 板式离心机(ELMI,型号:SkyLine CM-6MT)
  6. Glomax 96光度计(Promega,型号:GloMax 96)
    注意:本产品已停产。

程序

  1. 转染步骤
    注意:所有步骤都应在II类生物安全柜内进行,以避免污染。时间表:转染-24小时
    1. 293T / 17细胞应以转染时可提供70-90%汇合的比例传代培养至6孔板中。通常每孔接种4×10 5个细胞将达到这种融合水平。示例如图2所示

      图2. HEK293T / 17细胞转染前预期的融合实例

    时间表:转染日。
    1. 应使用水浴或孵化器将DMEM / 10%FBS / 1%P / S和Opti-MEM ®预热至37°C。
    2. 为将用于转染的6孔板的每个孔准备两个标记为无菌的1.5ml微量离心管(管1和管2)。
    3. 将以下用于转染的质粒构建体添加到管1:
      pCAGGS-SARS-CoV峰值:450ng
      p8.91-慢病毒载体:500ng
      pCSFLW:750 ng
    4. 向质粒DNA混合物(管1)中加入100μlOpti-MEM ®
    5. 向管2中加入100μlOpti-MEM 和17.5μl1mg / ml PEI。
    6. 孵育步骤:通过轻轻摇动混合两个管,并在室温(RT)下孵育5分钟
    7. 孵育后,将管2中的Opti-MEM / PEI溶液移至管1中的Opti-MEM ® / DNA溶液。
    8. 孵育步骤:在室温下孵育管20分钟,同时轻轻地将管子每3-4分钟混匀。
    9. 转染混合物孵化时,应去除293T / 17细胞上的培养基,每孔加入2ml新鲜的DMEM / 10%FBS / 1%P / S。在这一点上,必须缓慢地将培养基添加到井的一侧,以避免分离贴壁细胞单层。
    10. 孵育20分钟后,将DNA / Opti-MEM / PEI溶液移液至293T / 17细胞上,逐滴加入孔的整个表面积。轻轻旋转6孔板,以确保均匀分散的试剂混合物。
    11. 孵育步骤:将板在37℃,5%CO 2孵育过夜(o / n)。在我们手中,在12-16小时之间的孵化时间会导致相当的最终光伏产量RLU滴定度。
    时间轴:转染后12-16小时。
    1. 培养后培养细胞应小心取出,加入2ml新鲜的DMEM / 10%FBS / 1%P / S。再次将介质慢慢地添加到井的一侧,以避免细胞脱离
    2. 将6孔板在37℃孵育5%CO 2 / o / n 32-36小时。
    时间表:转染后44-52小时。
    1. 使用2.5ml无菌注射器收集含有病毒假型颗粒的上清液,随后通过注射器驱动的Millex HA-0.45μm过滤器过滤入Falcon或微量离心管。
    2. 将所有过滤的上清液储存在-80°C直到下游使用。建议将上清液等分储存,以避免可能影响病毒RLU滴度的多次冻融循环。
      注意:上清液可以在4℃下储存长达一周,没有可检测到的RLU滴度的损失。
    3. 任选步骤:另外的培养基可以添加到细胞中,以允许第二次收获18-24小时后,再加入DMEM / 10%FBS / 1%P / S。在这种情况下,在初始PV收集中必须格外小心(步骤A15),以避免损伤细胞单层。我们已经观察到,第一次收获后的健康状况不佳的细胞在第二次收获后导致PV生产的RLU滴度显着降低
    注意:可以通过遵循上述步骤创建控制假型病毒,但省略pCAGGS-SARS-CoV尖峰构建体。这产生不表达病毒表面糖蛋白的颗粒(Delta Env对照,图3)。

  1. 滴定步骤(图3)
    注意:滴定由报告人(在这种情况下是萤火虫萤光素酶)转导到由病毒假型(SARS-CoV尖峰)上表达的病毒糖蛋白介导的靶细胞中。用于滴定的控制是通过包含“仅细胞”和“Delta Env”列提供的。可以通过带有Vesicular口腔炎病毒G蛋白(VSV-G)的PV提供转导控制,该蛋白利用普遍存在的受体,在测试的所有细胞系中导致高RLU滴度。


    图3.用于假型滴定的96孔板装置串行稀释步骤显示向A列的每个孔中加入100μl假型病毒上清液,并从该孔中取出50μl的稀释液B.该过程继续到板(行H)的终点,此时最后的50μl被丢弃。 Delta Env控件用红色表示(第11列),单元格控件用绿色表示(第12列)。每个稀释系列(板)可以使用一套移液器吸头。

    1. 在96孔白板中,在“单元”对照的整列中加入50μl的DMEM / 10%FBS / 1%P / S(参见图3第12列)。
    2. 将50μlDMEM / 10%FBS / 1%P / S从B行添加到H,其中包含PV或Delta-Env对照病毒。
    3. 向A列(不包括对照色谱柱)的每个孔中加入100μlSARS假型病毒上清液,并加入100μlDelta-Env至第11列(见图3)。
    4. 从行A病毒的孔中取出50μl,并对其下面的所有孔进行两次连续稀释。
    5. 每个稀释步骤使用移液管混合8次,上下移动,注意不要产生气泡。
    6. 完成连续稀释后,应从每列最后一个孔中取出最终的50μl 注意:此时每个孔都应含有50μl混合的DMEM和PV上清液。
    7. 准备一片敏感靶细胞(Huh-7为SARS PV),优先传播1:4 48 h之前:
      1. 从板上取出培养基。
      2. 用2ml PBS洗板两次,弃去。
      3. 向板中加入2ml胰蛋白酶以分离细胞。
      4. 细胞分离后,向板中加入6ml DMEM / 10%FBS / 1%P / S以淬灭胰蛋白酶活性,并轻轻重悬细胞。
      5. 使用TC20 TM计数细胞/自动细胞计数器或血细胞计数器,并向每个孔中加入总共50μl的1×10 4个细胞。
    8. 离心板在800 x g(Rcf)下放置1分钟,如果孔两侧有可见的液滴。
    9. 在37℃5%CO 2 孵育板48小时。
    10. 在Glomax 96光度计(或等同物)上使用Bright Glo TM 荧光素酶测定系统读板。

  2. 基于假型的中和测定(pMN)
    注意:pMN是通过针对SARS糖蛋白的抗体(或抑制剂)抑制PV介导的转导。
    1. 在96孔白板中,向B列至H列1-12列加入50μlDMEM / 10%FBS / 1%P / S。
    2. 在100μlDMEM / 10%FBS / 1%P / S的总体积中,将已知量的抗体(例如图4中的5μlHN和R血清)加入到A列,2-10栏的孔中。向井A11和A12作为对照,加入已知量(例如,5μl)的阳性和阴性抗血清。


      图4.抗SARS抗体(感染后血清HN和R)中和SARS病毒假型进入Huh7细胞每个血清样品绘制两个重复。

    3. 从A排孔中取出50μl,并在其下面的所有孔上进行两次连续稀释。
    4. 每个稀释步骤,使用移液管,通过上下移动进行15次混合,注意不要产生气泡。
    5. 完成连续稀释后,应从每列最后一个孔中取出最终的50μl 注意:此时每个孔都应含有50μl混合的DMEM和连续稀释的抗体/抑制剂。
    6. 离心板在800 x g(Rcf)下1分钟,以确保在井壁上没有抑制剂或液体。
    7. 使用从滴定获得的数据,计算稀释您的SARS-PV所需的DMEM的量,以获得50μl中的1×10 6个/ uLU,总体积为5ml。例如,使用1×10 8 L / L的RLU / ml,应将1ml的PV与4ml的DMEM混合。
    8. 使用多通道移液管将此稀释的PV溶液混合,并将平板等分至每孔中,除了孔A6-A12(仅细胞控制)外。 A1-A6将仅用作病毒控制
    9. 离心板在800 x g(Rcf)1分钟,以确保没有病毒留在井壁上。
    10. 在37℃5%CO 2孵育板1小时,允许抗体/抑制剂结合SARS糖蛋白的时间。
    11. 准备一片敏感靶细胞(Huh-7为SARS PV),优先传播1:4 48 h之前:
      1. 从板上取出培养基。
      2. 用2ml PBS洗板两次,弃去。
      3. 向板中加入2ml胰蛋白酶以分离细胞。
      4. 细胞分离后,向板中加入6ml DMEM / 10%FBS / 1%P / S以淬灭胰蛋白酶活性,并轻轻重悬细胞。
      5. 使用TC20 TM计数细胞/自动细胞计数器或血细胞计数器,并向每个孔中加入总共50μl的1×10 4个细胞。
    12. 在37℃5%CO 2 孵育板48小时。
    13. 在Glomax 96光度计(或等同物)上使用Bright Glo TM 荧光素酶测定系统读板。

数据分析

  1. RLU读数通过每个孔的稀释因子(20x,40x,80x,160x,320x,640x,1,280x,2,560x)乘以RLU / ml。所有8 RLU / ml值的平均值用作滴定步骤中该柱所报告的最终值。在RLU值和PV稀释之间应该观察到线性关系,每稀释1:2后,值降低50%。在乘法之前应注意检查这种线性关系,因为尽管缺乏光伏或工作光伏,但这固有的可能导致误报(表1)。

    表1. PV滴定数据分析 RLU值乘以RLU / ml值(以绿色突出显示),给出每个稀释点的RLU / ml值。然后从所有8个稀释点计算平均值/平均值。必须注意观察稀释因子(X因子)和RLU之间的线性关系,或乘法可导致RLU / ml膨胀的值(例如黄色突出显示)。


  2. 方案读取和滴定结果(图5)

    图5.使用优化转染方案的假型产生RLU滴度的结果 SARS;具有SARS-CoV的PV表面上的尖峰,细胞;单元控制,三角洲环境;无表面糖蛋白,VSV-G;表面具有VSV-G的PV。

  3. 概述的方案提供了用于产生表达适合进一步下游R& D应用的SARS-CoV尖峰蛋白的高滴度病毒假型颗粒的快速且一致的方法。使用两种感染后血清(HN和R)对SARS-CoV假型病毒进行有效击倒(中和)显示了疫苗免疫原性和mAb /抗病毒筛选的潜在效用。使用容易获得的试剂应有助于提高再现性。

致谢

作者承认陈博士(香港中文大学医学院)两位阳性感染后控制血清。这项工作由Medway药学院,英国肯特资助。该协议由PMID修改和改进:15757556和26587388。

参考

  1. Carnell,GW,Ferrara,F.,Grehan,K.,Thompson,CP and Temperton,NJ(2015)。用于流感的基于假型的中和测定:系统分析。免疫组织 6:161.
  2. Grehan,K.,Ferrara,F.and Temperton,N。(2015)。用于产生MERS-CoV峰表达病毒假型的优化方法。方法X 13(2):379-384。
  3. Temperton,NJ(2009)。  使用逆转录病毒假型测定SARS冠状病毒的抗体应答。在Lal,SK(Ed。)。 SARS冠状病毒的分子生物学。 Springer 。
  4. Temperton,NJ,Chan,PK,Simmons,G.,Zambon,MC,Tedder,RS,Takeuchi,Y.and Weiss,RA(2005)。纵向表征中和抗体对SARS冠状病毒的假型的反应。 11(3) :411-416。
  5. Temperton,NJ,Wright,E.and Scott,SD(2015)。逆转录病毒假型 - 从科学工具到临床实用。 eLS 1-11。
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引用:Carnell, G., Grehan, K., Ferrara, F., Molesti, E. and Temperton, N. J. (2017). An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes. Bio-protocol 7(16): e2514. DOI: 10.21769/BioProtoc.2514.
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