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Detection of CLEC5A-JEV Interaction by ELISA
通过酶联免疫吸附法(ELISA)检测CLEC5A乙型脑炎病毒   

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Abstract

JEV (Japanese encephalitis virus) belonging to Flaiviridae interacts with CLEC5A (C-type lectin domain family 5, member A), a member of C-type lectin associated with DAP12 signaling protein and expressed on myeloid cell, as the same extent as Dengue virus. This protocol is used to perform and determine the interaction between purified JEV particles with human and murine CLEC5A.Fc fusion proteins by ELISA method. The different strain and batch of purified JEV as well as purity of CLEC5A.Fc fusion protein might affect the result of absorbance. Modifications are most likely needed if users intend to perform ELISA assay.

Keywords: CLEC5A(clec5a), Japanese encephalitis virus(日本脑炎病毒), Flavivirus(黄病毒), ELISA(ELISA)

Materials and Reagents

  1. The neurovirulent (RP-9) of Japanese encephalitis virus (JEV)
  2. Aedes albopictus mosquito cell line C6/36
  3. BHK21 cell line
  4. 20% sucrose
  5. Phosphate buffered saline (PBS)
  6. FreeStyle? 293-F cells (Life Technologies, Invitrogen™, catalog number: R790-07 )
  7. pcDNA3/CLEC5A_ECD.Fc plasmid (or plasmids carrying extracellular domain of other C-type lectin receptor)
  8. 293 fectin Transfection Reagent (Life Technologies, Invitrogen™, catalog number: 12347-019 )
  9. Gibco FreeStyle 293 Expression Medium (Life Technologies, Invitrogen™, catalog number: 12338018 )
  10. Protein A sepharose beads (GE Healthcare, catalog number: 17-0780-01 )
  11. HRP-conjugated anti-human IgG (Fc) (Jackson ImmunoResearch Laboratories, 109-035-003 )
  12. 3,3,5,5-tetramethylbenzidine (TMB) (BD Pharmingen, catalog number: 555214 )
  13. 1x RPMI-1640 (Life Technologies, Gibco®, catalog number: 31800-022 )
  14. Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 11091-148 )
  15. BSA (Sigma-Aldrich, catalog number: A2153 )
  16. KH2PO4
  17. Na2HPO4
  18. Blocking buffer (see Recipes)
  19. 1 L PBS buffer (see Recipes)

Equipment

  1. TECAN ELISA Reader
  2. Beckman Ultracentrifugation
  3. Pasteur pipette
  4. Centrifuge tubes (Corning Incorporated)
  5. Microtiter plate (Corning, Costar®, catalog number: 9018 )

Procedure

  1. Infected C6/36 cells (1 x 107) with JEV (M. O. I. = 0.1) for 2 h followed by removing virus and replenishing fresh RPMI 1640 medium containing 5% fetal bovine serum.
  2. After three days, supernatants were harvested and stocked; cells were further refilled with new fresh medium for another three days.
  3. Collect viral supernatant (10 ml/tube) and centrifuging supernatant at 8,000 rpm for 30 min at 4 °C.
  4. Add 10 ml of supernatant into centrifuge tube followed by 1 ml of adding 20% sucrose cushion at the bottom of centrifuge tube by using Pasteur pipette adding sucrose solution into bottom of the centrifuge tube.
  5. Centrifuge supernatant at 28,000 rpm (SW28) for 3.5 h at 4 °C.
  6. Remove supernatant and suspending the JEV particles in 0.5 ml PBS, and viral titer was determined by typical plaque assay using BHK21 cell line.
  7. 30 μg of pcDNA3/CLEC5A_ECD.Fc plasmid or extracellular domain of other C-type lectin receptor (CLR) plasmid were transfected into suspended FreeStyle? 293-F cells (3 × 107/30 ml) by using 30 μl of 293 fectin? Transfection Reagent.
  8. Supernatants were harvested at 48 h of post transfection and subjected to protein purification by protein A sepharose beads.
  9. For Fc.funsion prtoen purification, supernatants were passed through a protein A column [pre-equilibrated with 0.05 M Tris-HCl (0.1 M, pH 7.0 )], after binding, washed column with 20 ml of Tris-HCl (0.1 M, pH 7.0) and eluted with 4 ml of Tris-Glycine (0.1 M, pH 3.0) buffer.
  10. 50 μl of sucrose-cushion-purified JEV particles (5 × 106 pfu) in PBS were coated on microtiter plates for overnight at 4 °C, and washed with 250 μl of PBS for 3 times before adding CLR fusion proteins and control protein.
  11. Plate was filled with blocking buffer containing 1% BSA and 1% fetal bovine serum in PBS at room temperature for 1 h to reduce the detection background.
  12. Both human and murine CLEC5A.Fc fusion proteins (0.05 mg/ml in PBS; 100 μl/well) and hu-IgG1 control fusion proteins were added at the amount of 0.05 mg/ml; 100 μl /well in room temperature for 2 h to interact with coated JEV particle.
  13. Remove unbound JEV particle by gentle washing with 250 μl of PBS twice.
  14. HRP-conjugated anti-human IgG (Fc) (1:5,000) were used to detect fusion proteins for 1 h at room temperature followed by washing with 250 μl of PBS twice and using 100 μl of 3,3,5,5-tetramethylbenzidine (TMB) as substrate and detecting the absorbance at 450 nm in ELISA reader.

Recipes

  1. Blocking buffer
    1% BSA
    1% FBS
  2. 1 L PBS buffer (pH to 7.2-7.4)
    900 ml H2O
    8 g NaCl or 27.4 ml 5 M
    0.2 g KCl
    0.19 g KH2PO4 or 1.4 ml 1 M
    0.61 g Na2HPO4 or 4.3 ml 1 M

Acknowledgments

This protocol is adapted from Chen et al. (2012). We thank Drs. Caroline Milner and Carl Brown for critical comments. We are grateful for Dr. Tzyy-Wen Chiou, Dr. J-J Liang, Y-L. Lee, Chih-Ya Yang, Yu-Chiuan Wang and C-H, Chao for technical assistance. We also thank the Taiwan Mouse Clinic which is funded by the National Research Program for Genomic Medicine (NRPGM) at the National Science Council (NSC-99-3112-B-001-021) of Taiwan for technical support in histopathology experiments. We also acknowledge the technical services provided by the Transgenic Mouse Model Core Facility of the National Research Program for Genomic Medicine, NSC, and the technical services provided by Flow cytometry Core Facility of National Yang Ming University.

References

  1. Chen, S. T., Liu, R. S., Wu, M. F., Lin, Y. L., Chen, S. Y., Tan, D. T., Chou, T. Y., Tsai, I. S., Li, L. and Hsieh, S. L. (2012). CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality. PLoS Pathog 8(4): e1002655.
  2. Chen, S. T., Lin, Y. L., Huang, M. T., Wu, M. F., Cheng, S. C., Lei, H. Y., Lee, C. K., Chiou, T. W., Wong, C. H. and Hsieh, S. L. (2008). CLEC5A is critical for dengue-virus-induced lethal disease. Nature 453(7195): 672-676.

简介

属于黄病毒科的JEV( 病毒)与CLEC5A(C型凝集素结构域家族5,成员A) C型凝集素,与DAP12信号传导蛋白相关并且在骨髓细胞上表达,与登革热病毒的程度相同。 该方案用于通过ELISA方法进行和确定纯化的JEV颗粒与人和鼠CLEC5A.Fc融合蛋白之间的相互作用。 不同的菌株和批次的纯化JEV以及CLEC5A.Fc融合蛋白的纯度可能影响吸光度的结果。 如果用户打算进行ELISA测定,则很可能需要修饰。

关键字:clec5a, 日本脑炎病毒, 黄病毒, ELISA

材料和试剂

  1. 日本脑炎病毒(JEV)的神经毒性(RP-9)
  2. 白纹伊蚊蚊细胞系C6/36
  3. BHK21细胞系
  4. 20%蔗糖
  5. 磷酸盐缓冲盐水(PBS)
  6. FreeStyle? 293-F细胞(Life Technologies,Invitrogen TM,目录号:R790-07)
  7. pcDNA3/CLEC5A_ECD.Fc质粒(或携带其他C型凝集素受体的胞外结构域的质粒)
  8. 293 Fectin转染试剂(Life Technologies,Invitrogen TM,目录号:12347-019)
  9. Gibco FreeStyle 293表达培养基(Life Technologies,Invitrogen TM,目录号:12338018)
  10. 蛋白A琼脂糖珠(GE Healthcare,目录号:17-0780-01)
  11. HRP缀合的抗人IgG(Fc)(Jackson ImmunoResearch Laboratories,109-035-003)
  12. 3,3,5,5-四甲基联苯胺(TMB)(BD Pharmingen,目录号:555214)
  13. 1x RPMI-1640(Life Technologies,Gibco ,目录号:31800-022)
  14. 胎牛血清(FBS)(Life Technologies,Invitrogen TM,目录号:11091-148)
  15. BSA(Sigma-Aldrich,目录号:A2153)
  16. KH 2 PO 4
  17. Na 2 HPO 4
  18. 阻止缓冲区(参见配方)
  19. 1 L PBS缓冲液(见配方)

设备

  1. TECAN ELISA读数器
  2. Beckman超速离心
  3. 巴斯德移液器
  4. 离心管(Corning Incorporated)
  5. 微量滴定板(Corning,Costar ,目录号:9018)

程序

  1. 用JEV(M.O.I. = 0.1)感染C6/36细胞(1×10 7个/孔)2小时,然后除去病毒并补充含有5%胎牛血清的新鲜RPMI 1640培养基。
  2. 三天后,收获上清液并储备; 细胞再用新鲜的培养基再补充三天
  3. 收集病毒上清液(10ml /管),并在4℃下以8,000rpm离心上清液30分钟
  4. 加入10毫升的上清液到离心管,然后1毫升添加20%蔗糖垫在离心管的底部,使用巴斯德吸管添加蔗糖溶液到离心管的底部。
  5. 在4℃下以28,000rpm(SW28)离心上清液3.5小时
  6. 除去上清液并将JEV颗粒悬浮于0.5ml PBS中,通过使用BHK21细胞系的典型噬斑测定法测定病毒滴度。
  7. 将30μgpcDNA3/CLEC5A_ECD.Fc质粒或其他C型凝集素受体(CLR)质粒的胞外结构域转染入悬浮的FreeStyle? 293-F细胞(3×10 7/sup/30ml)。转染试剂
  8. 在转染后48小时收获上清液,并通过蛋白A琼脂糖珠粒进行蛋白质纯化
  9. 为了进行完全纯化,将上清液通过蛋白A柱[用0.05M Tris-HCl(0.1M,pH7.0)预平衡],结合后用20ml Tris-HCl(0.1M, pH 7.0) 用4ml Tris-甘氨酸(0.1M,pH 3.0)缓冲液洗脱
  10. 将50μl在PBS中的蔗糖缓冲液纯化的JEV颗粒(5×10 6 pfu)在4℃下在微量滴定板上包被过夜,并用250μlPBS洗涤3次,然后加入CLR融合蛋白和对照蛋白。
  11. 在室温下用含有1%BSA和1%胎牛血清的封闭缓冲液在PBS中填充板1小时以减少检测背景。
  12. 以0.05mg/ml的量加入人和鼠CLEC5A.Fc融合蛋白(0.05mg/ml,在PBS中;100μl/孔)和hu-IgG1对照融合蛋白;在室温下100μl/孔2小时以与涂布的JEV颗粒相互作用
  13. 通过用250μlPBS温和洗涤两次除去未结合的JEV颗粒
  14. 使用HRP-缀合的抗人IgG(Fc)(1:5,000)在室温下检测融合蛋白1小时,然后用250μlPBS洗涤两次,并使用100μl的3,3,5,5-四甲基联苯胺(TMB)作为底物,并在ELISA读数器中检测450nm处的吸光度

食谱

  1. 阻塞缓冲区
    1%BSA
    1%FBS
  2. 1 L PBS缓冲液(pH至7.2-7.4)
    900ml H 2 O 2 / 8克NaCl或27.4毫升5毫升 0.2克KCl
    0.19g KH 2 PO 4 4或1.4ml 1M w/v 0.61g Na 2 HPO 4或4.3ml 1M

致谢

该协议改编自Chen等人(2012)。我们感谢博士。 Caroline Milner和Carl Brown对于批评性评论。我们感谢Tzyy-Wen Chiou博士,J-J Liang博士,Y-L。 Lee,Chih-Ya Yang,Yu-Chiuan Wang和C-H,Chao提供技术帮助。我们还要感谢由台湾国家科学委员会(NSC-99-3112-B-001-021)的国家基因组医学研究计划(NRPGM)资助的台湾小鼠诊所,在组织病理学实验中提供技术支持。我们还承认由国家基因组医学研究计划,NSC的转基因小鼠模型核心设施提供的技术服务,以及由阳明大学流式细胞术核心设施提供的技术服务。

参考文献

  1. Chen,S.T.,Liu,R.S.,Wu,M.F.,Lin,Y.L.,Chen,S.Y.,Tan,  Chou,T.Y.,Tsai,I.S.,Li,L.and Hsieh,S.L。(2012)。 CLEC5A可调节日本脑炎病毒诱导的神经炎症和致死率 PLoS Pathog 8(4):e1002655。
  2. Chen,S.T.,Lin,Y.L.,Huang,M.T.,Wu,M.F.,Cheng,S.C.,Lei,H.Y.,Lee,C.K.,Chiou,T.W.,Wong,C.H.and Hsieh, CLEC5A对于登革热病毒诱导的致死性疾病至关重要。 /em> 453(7195):672-676。
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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。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, S. and Hsieh, S. E. (2012). Detection of CLEC5A-JEV Interaction by ELISA. Bio-protocol 2(19): e268. DOI: 10.21769/BioProtoc.268.
  2. Chen, S. T., Liu, R. S., Wu, M. F., Lin, Y. L., Chen, S. Y., Tan, D. T., Chou, T. Y., Tsai, I. S., Li, L. and Hsieh, S. L. (2012). CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality. PLoS Pathog 8(4): e1002655.
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