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Sep 2019
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Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus
基于分割纳米荧光素酶的乙型脑炎病毒组装检测   

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Abstract

Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such analyses. We developed a method for viral particle analysis that integrates a high-sensitivity split luciferase system and density gradient centrifugation. This protocol enables high-resolution profiling of particles produced by cells expressing Japanese encephalitis virus factors.

Keywords: Flavivirus (黄病毒), Japanese encephalitis virus (乙型脑炎病毒), Virion assembly (病毒颗粒组装), HiBiT tag (HiBiT标签), Split nano luciferase (分割纳米荧光素酶)

Background

The flavivirus, a group of arboviruses including dengue virus, West Nile virus, tick-borne encephalitis virus, Zika virus, yellow fever virus, and Japanese encephalitis virus (JEV), is associated with substantial morbidity and mortality in human populations (Chambers et al., 1990). Flaviviruses have a single ORF genome encoding a long polypeptide, which is post-translationally cleaved into three structural (C, prM and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins by the host signal peptidase and NS2B-NS3 viral protease (Chambers et al., 1990).

Viral genomic RNA synthesized by the NS5 RNA-dependent RNA polymerase associates with C protein to form the nucleocapsid, which in turn is packaged in the envelope composed of host cell-derived lipid membrane, and viral prM and E transmembrane proteins to form the virion. It has been found that a reduced form of flaviviral genomic RNA, designated as subgenomic replicon, which lacks all structural genes, can replicate itself in host cells (Suzuki et al., 2014), and that the subgenomic RNA is incorporated into the virus-like particle to form single-round infectious particles (SRIPs) when C, prM and E proteins are supplied in trans (Suzuki et al., 2014; Matsuda et al., 2018).

Besides major infectious particles, virus-infected cells often produce minor particles, some of which are hard to detect with immunochemical methods, due to their small amounts. To study typical and atypical particles released from flavivirus-infected cells, we modified a SRIP system (Matsuda et al., 2018) to develop a JEV HiBiT-SRIP assay system. In this system, the C protein is labeled with a highly sensitive HiBiT peptide tag, to enable high-resolution sedimentation profiling of particles produced by the cells expressing viral structural and nonstructural factors (Ishida et al., 2019). Cells transfected with a set of plasmids (referred to as SRIPs-producer cells) produce SRIPs and non-infectious subviral particles containing C-HiBiT, which can be separated by sucrose density gradient centrifugation. SRIPs in sedimentation fractions can be detected by measurement of cellular NanoLuc levels after inoculation of other cells (referred to as target cells) with all the fractions, which allows SRIP-borne NanoLuc expression (Figure 1). It is possible to apply the principle of JEV HiBiT-SRIP assay to other viruses.


Figure 1. Overview of HiBiT-SRIP system. A. HiBiT-SRIP system plasmids. HDV-Rz: self-cleaving HDV Ribozyme; pA: SV40 polyadenylation signal; CMVp: CMV promoter; CAGp: CAG promoter. B. Schematic presentation of HiBiT-SRIP experiments. JErep-nluc: subgenomic replicon with a gene for NanoLuc as the reporter.

Materials and Reagents

  1. 1.5 ml tube (Watson, catalog number: 131-815C )
  2. 2 ml tube (Watson, catalog number: 132-620C )
  3. 6-well cell culture plate (Violamo, catalog number: 2-8588-01 )
  4. 96-well cell culture plate (Violamo, catalog number: 2-8588-05 )
  5. 14 x 95 mm open-top polyclear centrifuge tubes (Seton Scientific, catalog number: 7031 )
  6. White 384-well immuno plates (Thermo, catalog number: 460372 )
  7. 0.22 μm filter (Merck Millipore, catalog number: UFC30GV00 )
  8. 293T cells (ATCC, catalog number: CRL-3216 )
  9. Huh7 cells (JCRB, catalog number: JCRB0403 )
  10. Plasmid pCAG-JEC-FLAG-HiBiT which encodes HiBiT-tagged wild-type JEV C protein (Ishida et al., 2019; available from authors upon request) or its variant encoding mutant forms of C protein
  11. Plasmid pCAG-prME which encodes JEV prM and E proteins (Suzuki et al., 2014; available from authors upon request)
  12. Plasmid pCMV-JErep-nluc which expresses a subgenomic replicon RNA of JEV, which contains a NanoLuc reporter gene (Matsuda et al., 2018, Ishida et al., 2019; available from authors upon request)
  13. Nano Glo HiBiT lytic detection system (Promega, catalog number: N3040 )
  14. Nano-Glo luciferase assay system (Promega, catalog number: N1120 )
  15. Dulbecco’s modified Eagle’s media (Nacalai, catalog number: 08458-16 )
  16. FBS (fetal bovine serum) (Thermo, catalog number: 10270-106 )
  17. PBS (phosphate-buffered saline without calcium and magnesium) (Nacalai, catalog number: 14249-24 )
  18. Benzylpenicillin potassium (Fujifilm, catalog number: 021-07732 )
  19. Streptomycin sulfate (Tokyo Chemical Industry, catalog number: S0585 )
  20. PEI MAX (Polyscience, catalog number: 24765-1 ), a transfection reagent
  21. Opti-MEM (Thermo, catalog number: 31985062 ), reduced serum medium which allows to keep high transfection efficiency with less cytotoxicity
  22. Sucrose, centrifugal density-gradient grade (Nacalai, catalog number: 30406-25 )
  23. Tris (Tris (hydroxymethyl) aminomethane) (Nacalai, catalog number: 35406-91 )
  24. NaCl (Nacalai, catalog number: 31320-05 )
  25. Triton X-100 (Nacalai, catalog number: 35501-15 )
  26. KCl (Nacalai, catalog number: 28513-85 )
  27. Na2HPO4 (Nacalai, catalog number: 31726-05 )
  28. KH2PO4 (Nacalai, catalog number: 28720-65 )
  29. PEI solution (1 mg/ml) (see Recipes)
  30. 100x penicillin G + streptomycin stock solution (see Recipes)
  31. Culture media (see Recipes)
  32. 10x PBS (pH 7.4) (see Recipes)
  33. 10% sucrose/PBS (see Recipes)
  34. 45% sucrose/PBS (see Recipes)
  35. 10-45% sucrose linear gradient/PBS (see Recipes)
  36. Lysis buffer (see Recipes)

Equipment

  1. Humidified incubator (PHCBI, model: MCO-170AICUVD-PJ , 37 °C, 5% CO2)
  2. Ultracentrifuge (Hitachi, model: CP80NX )
  3. Swing bucket rotor (Hitachi, model: P40ST )
  4. Gradient mixer (BIOCOMP, Gradient Mate)
  5. Piston gradient fractionator (BIOCOMP, catalog number: 153 )
  6. Microplate luminometer (Thermo, Varioskan LUX Multimode Microplate Reader)
  7. -30 °C freezer (PHCBI, catalog number: MDF-MU539D )

Procedure

Flow of following procedures is presented in Figure 2.



Figure 2. Flow of procedures


  1. Production of HiBiT-containing particles
    1. Seed 293T cells in 6-well plates, at density of ~50,000 cells/well in 2 ml growth medium.
    2. Place the plates in a CO2 incubator for 24 h.
    3. Mix 0.2 µg pCAG-JEC-HiBiT, 0.4 µg pCMV-JErep-nluc and 0.2 µg pCAG-JEprME in 200 µl Opti-MEM, mix well with a vortex mixer.
      (Optional) Set control experiments using an empty vector in place of each plasmid.
      (Optional) Set experiments using plasmids encoding mutant forms of viral elements of interest.
    4. Add 2.4 µl PEI solution (1 mg/ml) to the mixture and mix well with a vortex mixer.
    5. Incubate the DNA-PEI mixture for 15 min at room temperature.
    6. Apply the DNA-PEI mixture to cell-containing wells (~200 μl/well).
    7. Place the plates in a CO2 incubator.
    8. Replenish growth media 6 h post-transfection.
      Note: Add media slowly to the side of the well to avoid cell detachment.
    9. Replenish growth media 48 h post-transfection.
      Note: Add media slowly to the side of the well to avoid cell detachment.
    10. Transfer the culture supernatant 72 h post-transfection to 2 ml tubes.
    11. Centrifuge the tube at 20,000 x g, 4 °C for 10 min.
    12. Transfer supernatant to fresh tubes and proceed to Procedure B or Procedure C.

  2. Evaluation of replication efficiency of the subgenomic replicon RNA in SRIPs-producer cells (a typical result is presented in Figure 2A)
    1. Suspend cells in each well in 1 ml PBS and transfer to a 1.5 ml tube.
    2. Centrifuge the tubes at 2,400 x g, 4 °C for 5 min.
    3. Aspirate the supernatant, add 50 μl lysis buffer and mix by pipetting.
    4. Centrifuge the tubes at 20,000 x g, 4 °C for 10 min.
    5. Transfer 5 μl of the clear lysate in each tube to 384-well plate.
    6. Add 5 μl of NanoLuc substrate diluted 1:600 with lysis buffer.
    7. Incubate for 10 min at room temperature.
    8. Measure the luminescence using a microplate reader.

  3. Titration of SRIPs in culture supernatant (a typical result is presented in Figure 2C)
    1. Seed Huh7 cells in 96-well plates, at density of ~10,000 cells/well and incubate in a CO2 incubator overnight.
    2. Inoculate 100 μl of culture supernatant (prepared with Procedure B) into each well.
    3. Incubate for 72 h.
    4. Aspirate growth media and add 50 μl lysis buffer to each well and mix by pipetting.
    5. Transfer 5 μl of the lysate in each well to a 384-well plate.
    6. Add 5 µl of NanoLuc substrate diluted 1:600 with lysis buffer.
    7. Incubate for 10 min at room temperature.
    8. Measure the luminescence using a microplate reader.

  4. Sucrose density gradient centrifugation of the culture supernatant
    1. Layer 300 μl of the culture supernatant containing HiBiT-labeled particles on continuous 10-45% sucrose gradient/PBS in Open-Top Polyclear centrifuge tubes (SETON, 14 x 95 mm).
    2. Centrifuge the gradients at 100,000 x g for 3 h at 4 °C with a pre-chilled swing bucket rotor.
    3. Fractionate the gradients from top to bottom (total < 40 fraction), with a piston fractionator (2.3 mm/fraction, 0.3 mm/s) to a 96-well plate.

  5. Titration of C-HiBiT-containing particles in sedimentation fractions (a typical result is presented in Figure 2B)
    1. Transfer 5 μl of the fraction in each well to 384-well plate.
    2. Add 5 μl of HiBiT reagent to each well.
    3. Incubate for 10 min at room temperature.
    4. Measure the luminescence using a microplate reader. Typical results out of 3 > experiments are indicated in the figures.

  6. Titration of SRIPs in sedimentation fractions (a typical result is presented in Figure 2D)
    1. Seed Huh7 cells in 96-well plates, at density of ~10,000 cells/well and incubate in a CO2 incubator overnight.
    2. Inoculate 5 μl of sedimentation fractions to each well.
    3. Incubate for 72 h.
    4. Aspirate growth media and add 50 μl lysis buffer to each well and mix by pipetting.
    5. Transfer 5 μl of the lysate in each well to 384-well plate.
    6. Add 5 µl of NanoLuc substrate diluted 600 fold with lysis buffer.
    7. Incubate for 10 min at room temperature.
    8. Measure the luminescence using a microplate reader (Figure 3).


      Figure 3. Typical results. (A) NanoLuc activity of producer cell lysate to evaluate autonomous replication of the subgenomic RNA (B) HiBiT activities of fractions of producer cell culture supernatant, fractionated via sucrose density gradient centrifugation. (C) NanoLuc activity of target cells inoculated with producer cell supernatant, which reflects the infectious titer of SRIPs in the supernatant. (D) NanoLuc activities of target cells inoculated with sucrose density gradient centrifugation fractions of producer cell supernatant, which reflect the infectious titer of SRIPs in the fractions.

Notes

  1. Strong luminescence emitted by NanoLuc/HiBiT system sometimes leaks to adjacent wells in 384-well plates. Therefore, the recommendation is to load samples into every other well. In addition, sometimes it is better to dilute lysates or fractions which produce strong signals to minimize leakage of luminescence to neighboring wells (Procedures B, D, E and F).
  2. Be aware that it is difficult to estimate relative amounts of C-HiBiT in top fractions when titrating HiBiT-SRIPs in sedimentation fractions (Procedure B), because free NanoLuc proteins that leak from cells in the fractions also contribute to signal production.

Recipes

  1. PEI solution (1 mg/ml)
    PEI MAX 100 mg
    ddH2O up to 100 ml
    Sterilize via a 0.22 μm filter, aliquot to 15 ml tubes and keep a tube as a working stock in a refrigerator and the rest of the tubes in a -30 °C freezer
  2. 100x penicillin G + streptomycin stock solution
    PBS 100 ml
    Benzylpenicillin potassium 0.626 g
    Streptomycin sulfate 1 g
    Sterilize via a 0.22 μm filter and store in a refrigerator
  3. Culture media
    Dulbecco’s modified Eagle’s medium 500 ml
    FBS, heat-inactivated by incubation at 56 °C for 45 min 50 ml
    100x penicillin G + streptomycin stock solution 5 ml
  4. 10x PBS (pH 7.4)
    NaCl 80 g
    KCl 2.0 g
    Na2HPO4 14.4 g
    KH2PO4 2.4 g
    ddH2O up to 1 L
  5. 10% sucrose/PBS
    Sucrose 100 g
    10x PBS 100 ml
    ddH2O up to 1 L
    Sterilize via a 0.22 μm filter and store at room temperature
  6. 45% sucrose/PBS
    Sucrose 450 g
    10x PBS 100 ml
    ddH2O up to 1 L
    Sterilize via a 0.22 μm filter and store at room temperature
  7. 10-45% sucrose linear gradient/PBS
    Pour appropriate volume of 10% sucrose/PBS to 14 x 95 mm open-top polyclear centrifuge tubes, underlay 45% sucrose/PBS from bottom of the tubes using a needle attached syringe. Form linear gradients using gradient former with following parameters:
    Time: 2 min 5 s
    Angle: 82°
    Speed: 16 rpm
    After forming the gradients, chill them by standing several h at 4 °C
  8. Lysis buffer
    1 M Tris pH 7.5 20 ml
    5 M NaCl 30 ml
    Triton X-100 10.7 g
    ddH2O up to 1 L

Acknowledgments

We thank Jiahui Ong for proofreading the manuscript. We thank Mami Matsuda for the helpful advice and suggestions to develop HiBiT-SRIP system.

Competing interests

The authors have no competing interests directly relevant to the content of this article.

References

  1. Chambers, T. J., Hahn, C. S., Galler, R. and Rice, C. M. (1990). Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44: 649-688.
  2. Ishida, K., Goto, S., Ishimura, M., Amanuma, M., Hara, Y., Suzuki, R., Katoh, K. and Morita, E. (2019). Functional correlation between subcellular localizations of japanese encephalitis virus capsid protein and virus production. J Virol 93(19).
  3. Matsuda, M., Yamanaka, A., Yato, K., Yoshii, K., Watashi, K., Aizaki, H., Konishi, E., Takasaki, T., Kato, T., Muramatsu, M., Wakita, T. and Suzuki, R. (2018). High-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type 1 reporter replicon. Sci Rep 8(1): 16624.
  4. Suzuki, R., Ishikawa, T., Konishi, E., Matsuda, M., Watashi, K., Aizaki, H., Takasaki, T. and Wakita, T. (2014). Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon. J Gen Virol 95(Pt 1): 60-65.

简介

[ 摘要] 黄病毒感染的细胞释放出各种形式的传染性和非传染性颗粒,分别作为产物和副产物。通过密度梯度离心对释放的颗粒进行全面分析,有助于理解病毒颗粒的组装,但是很难检测到低水平的病毒颗粒。我们开发了一种将高灵敏度分裂荧光素酶系统和密度梯度离心相结合的病毒颗粒分析方法,该协议可对表达日本脑炎病毒因子的细胞产生的颗粒进行高分辨率分析。

[ 背景 ] 黄病毒是包括病毒,登革热病毒,西尼罗河病毒,tick传脑炎病毒,寨卡病毒,黄热病病毒和日本脑炎病毒(JEV)在内的一组虫媒病毒,与人类的大量发病和死亡相关( Chambers 等人,1990)。黄病毒具有编码长多肽的单个ORF基因组,该ORF基因组在翻译后被切割成三个结构性(C,prM 和E)和七个非结构性(NS1,NS2A,NS2B,NS3,NS4A)。 ,NS4B和NS5)蛋白通过宿主信号肽酶和NS2B-NS3病毒蛋白酶(Chambers 等,1990)。

由NS5 RNA依赖的RNA聚合酶合成的病毒基因组RNA与C蛋白结合形成核衣壳,然后将其包装在由宿主细胞衍生的脂质膜,病毒prM 和E跨膜蛋白组成的包膜中以形成病毒体。已经发现,缺乏所有结构基因的还原型黄病毒基因组RNA被称为亚基因组复制子,可以在宿主细胞中自我复制(Suzuki 等,2014),并且亚基因组RNA被整合入病毒中。当C,prM 和E蛋白反式供应时,类似颗粒的颗粒会形成单轮传染性颗粒(SRIP)(Suzuki 等,2014; Matsuda 等,2018)。

除了主要的感染性颗粒外,受病毒感染的细胞通常还会产生较小的颗粒,由于其数量很少,因此很难用免疫化学方法检测到。为了研究从黄病毒感染的细胞中释放出的典型和非典型颗粒,我们对SRIP系统进行了改进( Matsuda 等人,2018)开发了JEV HiBiT -SRIP分析系统,在该系统中,C蛋白被高敏感的HiBiT 肽标签标记,从而能够对表达病毒结构的细胞产生的颗粒进行高分辨率的沉淀分析非结构因素和(石田等,2019)。细胞转染了一组质粒(简称SRIPs生成细胞)产生SRIPs和非感染性亚病毒颗粒含C- HiBiT ,它可以通过分离蔗糖密度梯度离心沉淀级分中的SRIP 可以通过在所有级分whi接种到其他细胞(称为靶细胞)后测量细胞NanoLuc 水平来检测。ch允许SRIP携带的NanoLuc 表达(图1)。可以将JEV HiBiT -SRIP分析的原理应用于其他病毒。



D:\重新格式化\ 2020-3-2 \ 1902991--1375森田英治812018 \ Figs jpg \ Fig1.jpg

1.图的概述HiBiT -SRIP系统。阿。HiBiT -SRIP系统质粒HDV-RZ:自切割HDV核酶; pA的:SV40聚腺苷酸化信号; CMVP :CMV启动子的启动子; CAGP :CAG启动子启动子B中。。的概略presentation.dll HiBiT -SRIP实验。JErep-nluc :亚基因组复制子,带有NanoLuc 基因作为报告基因。

关键字:黄病毒, 乙型脑炎病毒, 病毒颗粒组装, HiBiT标签, 分割纳米荧光素酶

材料和试剂


 


1. 1.5ml试管(沃森,目录号的:131-815C)      


2. 2毫升管(沃森,目录号的:132-620C)      


3. 6孔细胞培养板(Violamo ,目录号:2-8588-01)      


4. 96孔细胞培养板(Violamo ,目录号:2-8588-05)      


5. 14×95毫米开顶Polyclear 离心管(西顿科学,目录编号:7031)      


6. 白色的384孔免疫板(Thermo ,目录号:460372)      


7. 0.22 Myuemu 过滤器(默克密理博,目录号:UFC30GV00)      


8. 293T细胞(ATCC ,目录号:CRL-3216)      


9. Huh7细胞(JCRB ,目录号:JCRB0403)      


10. 质粒PCAG -JEC-FLAG- HiBiT 编码HiBiT -标记的野生型JEV C蛋白(石田等人,2019;可从作者根据要求)ö ř及其变体编码突变晶型C蛋白的   


11. 编码JEV prM 和E蛋白的质粒pCAG-prME (Suzuki 等,2014;应要求可从作者处获得)   


12. 质粒PCMV-JErep-Nluc 其表达Subgeno 麦克风复制子RNA的JEV,其中包含一个NanoLuc 报道基因(松田等人,2018年,石田等人,2019;可从作者根据要求)   


13. Nano Glo HiBiT 裂解检测系统(Promega,目录号:N3040)   


14. Nano-Glo荧光素酶测定系统(Promega ,目录号:N1120)   


15. Dulbecco改良的Eagle媒体(Nacalai ,目录号:08458-16)   


16. FBS(胎牛血清)(Thermo,目录号:10270-106)   


17. PBS(磷酸盐缓冲液不含钙和镁)(ナ,目录号:14249-24)   


18. 苄青霉素钾(Fujifilm,目录号:021-07732)   


19. 硫酸链霉素(Tokyo Chemical Indus try,目录号:S0585)   


20. PEI MAX(PolyScience产品,目录号:24765-1) ,转染试剂   


21. Opti-MEM(Thermo,目录号:31985062),减少的血清培养基,可保持较高的转染效率,且细胞毒性较小   


22. 蔗糖,离心密度梯度等级(Nacalai ,目录号:30406-25)   


23. Tris (Tris(羟甲基)氨基甲烷)(Nacalai ,目录号:35406-91)   


24. NaCl (Nacalai ,目录号:31320-05)   


25. Triton X-100(Nacalai ,目录号:35501-15)   


26. KCl (Nacalai ,目录号:28513-85)   


27. 娜2 HPO 4 (ナ,目录号码:31726-05)   


28. KH 2 PO 4 (Nacalai ,目录号:28720-65)   


29. PEI溶液(1毫克/毫升)(请参阅食谱)   


30. 100x青霉素G +链霉素原液(请参阅食谱)   


31. 文化媒体(请参阅食谱)   


32. 10X PBS(pH 7.4)中(见配方)   


33. 10%蔗糖/ PBS(请参阅食谱)   


34. 45%蔗糖/ PBS(请参阅食谱)   


35. 10-45%蔗糖线性梯度/ PBS(请参阅食谱)   


36. 裂解缓冲液(请参见食谱)   


 


配套设备


 


加湿培养箱(PH CBI,型号:MCO-170AICUVD-PJ,37°C,5%CO 2 )
超速离心机(日立,型号:CP80NX)
摆斗式转子(日立,型号:P40ST)
梯度混合器(BIOCOMP,梯度配合)
活塞梯度分馏仪(BIOCOMP,目录号:153)
微孔板光度计(Thermo ,Varioskan LUX多模式微孔板读取器)
-30 °C冷冻室(PHCBI,目录号:MDF-MU539D)
程序


 


以下过程的流程如图2所示。


 


D:\重新格式化\ 2020-3-2 \ 1902991--1375森田英治812018 \ Figs jpg \ Fig2.jpg


˚F Igure 2.流量程序


 


含HiBiT 的颗粒的生产
将293T细胞接种到6孔板中,并在2 ml生长培养基中以〜50,000个细胞/孔的密度接种。
将板放在CO 2 培养箱中24小时。
0.2 Myug混合PCAG -JEC- HiBiT ,0.4 Myug PCMV-JErep-Nluc 和0.2 Myug PCAG-JEprME 在200 Myueru的Opti-MEM,拌匀用涡旋搅拌机。
(可选)小号的Et控制实验使用空载体代替每种质粒。


(可选)小号的Et实验使用的质粒编码突变形式的病毒元件兴趣。


向混合物中加入2.4 µl PEI溶液(1 mg / ml),并用涡旋混合器充分混合。
在室温下将DNA-PEI混合物孵育15分钟。
将DNA-PEI混合物施加到含细胞的孔中(〜200μl / 孔)。
将板放在CO 2 培养箱中。
转染后6小时补充生长培养基。
注意:将培养基缓慢加入孔的侧面,以避免细胞分离。


转染后48小时补充生长培养基。
注意:将培养基缓慢加入孔的侧面,以避免细胞分离。


转染后72小时将培养上清液转移至2 ml管中。
在20,000 xg ,4 °C下离心10分钟。
将吸管转移到新鲜的试管中,然后进行步骤B或步骤C。
 


评估SRIPs产生细胞中亚基因组复制子RNA 的复制效率(典型结果如图2A所示)。
将每个孔中的细胞悬浮于1 ml PBS中,并转移至1.5 ml管中。
将离心管在2,400 xg ,4°C下离心5分钟。
吸出补品,添加50μl 裂解缓冲液并通过移液混合。
在20,000 xg ,4°C下离心10分钟。
5传输Myueru 的澄清的裂解每管384 - 孔板。
加入5μl 用裂解液以1:600稀释的NanoLuc 底物。
在室温下孵育10分钟。
使用酶标仪测量发光。
 


培养上清液中SRIP的滴定(典型结果如图2C所示)。
将Huh7细胞以〜10,000个细胞/孔的密度播种到96孔板中,并在CO 2 培养箱中孵育过夜。
在每个孔中接种100μl 培养上清液(用步骤B制备)。
孵育72小时。
吸出生长培养基,向每个孔中加入50μl 裂解缓冲液,然后通过移液混合。
将每个孔中的5μl 裂解物转移到384孔板中。
加入5 µl 用裂解缓冲液稀释为1:600 的NanoLuc 底物。
在室温下孵育10分钟。
使用酶标仪测量发光。
 


培养液的蔗糖密度梯度离心
第3层00 Myueru 的培养上清含HiBiT 标记的颗粒在连续10-45 Pasento蔗糖梯度/ PBS在开顶Polyclear 离心管(SETON,14 X 95 毫米)。
使用预冷的回转铲斗转子在4 °C下以100,000 xg 的速度离心3小时。
梯度从分馏从上到下(总计< 40分),与活塞分馏(2.3毫米/级分,0.3毫米/秒)到96 - 孔平板中。   
 


沉淀级分中含C- HiBiT 的颗粒的滴定(典型结果如图2B所示)。
5传输Myueru 的分数在每个孔中以384 - 孔板。
5加入Myueru 中HiBiT 试剂,每孔。
在室温下孵育10分钟。
使用酶标仪测量发光度.3>实验中的典型结果显示在图中。
 


沉淀物中SRIP的滴定(典型结果如图2D所示)。
将Huh7细胞以〜10,000个细胞/孔的密度播种到96孔板中,并在CO 2 培养箱中孵育过夜。
在每个孔中接种5μl 沉淀级分。
孵育72小时。
吸出生长培养基,向每个孔中加入50μl 裂解缓冲液,然后通过移液混合。
5传输Myueru 裂解液中。每孔384 - 孔板。
加入5μl 用裂解缓冲液稀释600倍的NanoLuc 底物。
在室温下孵育10分钟。
发光使用测量微孔板读尔(图3) 。
 


D:\重新格式化\ 2020-3-2 \ 1902991--1375森田英治812018 \ Figs jpg \ Fig3.jpg


3.典型的结果图。(A)NanoLuc 活性的生产细胞裂解物,以评估中自主复制的亚基因组RNA(B)HiBiT 活动馏分的生产细胞培养物上清液,分馏通过蔗糖密度梯度离心。(C)NanoLuc 活性的目标(D)用生产者细胞上清液的蔗糖密度梯度离心分离级分接种的靶细胞的NanoLuc 活性,其反映了级分中SRIP的感染性滴度。


 


注意事项


 


NanoLuc / HiBiT 系统发出的强发光有时会泄漏到384 孔板的相邻孔中,因此建议将样品加载到其他每个孔中,此外,有时最好稀释能产生强信号的裂解物或馏分以将其最小化发光泄漏到邻近的孔(过程B,D,E和F)。
请注意,在滴定级分中的HiBiT -SRIPs 滴定时,很难估算顶部级分中C- HiBiT的相对含量(步骤B),因为从级分中的细胞泄漏的游离NanoLuc 蛋白也有助于信号产生。
 


菜谱


 


PEI溶液(1 毫克/毫升)
PEI最大100毫克             


ddH 2 O 高达100 ml             


通过A 0.22灭菌器Myuemu 过滤,分装至15ml管中并保持管作为工作原液在冰箱中,其余的管子在一个-30 ℃的冰箱中


100x青霉素G +链霉素原液
PBS 100毫升                                                       


苄青霉素钾0.626克             


硫酸链霉素1克                           


通过0.22灭菌器Myuemu 费尔之三和储存在冰箱


文化媒体
Dulbecco改良的Eagle培养基500毫升                                                       


FBS,通过在56°C下孵育45分钟50 ml 进行热灭活             


100x青霉素G +链霉素原液5毫升                           


10x PBS(pH 7.4)
氯化钠80克             


氯化钾2.0克                           


Na 2 HPO 4 14.4克             


KH 2 PO 4 2.4克             


ddH 2 O 至1 L             


10%蔗糖/ PBS
蔗糖100克             


10x PBS 100毫升             


ddH 2 O 至1 L             


通过0.22灭菌器Myuemu 过滤接- R和室温保存


45%蔗糖/ PBS
蔗糖450克             


10x PBS 100毫升             


ddH 2 O 至1 L             


通过0.22灭菌器Myuemu 过滤接- R和室温保存


1 0 -45 Pasento SUCRO 硒线性渐变/ PB 小号
将适量的10%蔗糖/ PBS倒入14 x 95 mm敞开式多透明离心管中,使用针头注射器在管的底部覆盖45%蔗糖/ PBS,使用具有以下参数的梯度形成剂形成线性梯度:


时间:2分钟5 s                           


甲Ngle:82 °             


小号皮德:16的rpm             


形成梯度后,将其在4°C放置几小时以使其冷却


裂解缓冲液
1 M Tris pH 7.5 20毫升             


5 M氯化钠30毫升                           


海卫一X-100 10.7克             


ddH 2 O 至1 L                           


 


致谢


 


谢谢我们嘉汇翁校对手稿。我们感谢松田麻美为有用的意见和建议,制定HiBiT -SRIP系统。


 


竞争利益


 


作者没有与本文内容直接相关的竞争利益。


 


[R Eferences


 


Chambers,TJ,Hahn,CS,Galler,R。和Rice,CM(1990),黄病毒基因组的组织,表达和复制, Annu Rev Microbiol 44:649-688。
Ishida,K.,Goto,S.,Ishimura,M.,Amanuma,M.,Hara,Y.,Suzuki,R.,Katoh,K. and Morita,E.(2019)。日本人亚细胞定位之间的功能相关性脑炎病毒衣壳蛋白和病毒生产,《病毒学杂志》93(19)。
松田(Matsuda),山中(Yamanaka),安藤(Yato),吉井(Yoshii),渡边(Washi),K.Aizaki,H。,小西(Konishi),高崎(T.),加藤(T.),村松(M.), Wakita,T.和Suzuki,R.(2018)。使用登革热1型报告基因复制子,基于单轮感染颗粒对多种黄病毒进行高通量中和测定。科学报告8(1):16624。
R. Suzuki,R. Ishikawa,T. Konishi,E.,Matsuda,M.,Watashi,K.,Aizaki,H.,Takasaki,T.和Wakita,T.(2014)。单轮传染性嵌合体的生产。带有基于DNA的日本脑炎病毒复制子的黄病毒, J Gen Virol 95(Pt 1):60-65。
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引用:Goto, S., Ishida, K., Suzuki, R. and Morita, E. (2020). Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus. Bio-protocol 10(9): e3606. DOI: 10.21769/BioProtoc.3606.
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