JoVE Journal > Immunology and Infection
Summary
Abstract
Protocol
Discussion
Declarações
Acknowledgements
Materials
Referências
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Imunologia e Infecção

表面等离子体共振检测到宿主细胞质的毒素易位

Published: January 03, 2012
doi:
10.3791/3686
, , ,
1Department of Molecular Biology and Microbiology,University of Central Florida

Summary

在这份报告中，我们描述了表面等离子体共振是如何用来检测输入到宿主细胞质中的毒素。胞浆内毒素量，这个高度敏感的方法可以提供定量数据，并可以应用到一系列的毒素。

Abstract

AB毒素组成的酶的A亚单位和一个细胞结合B 亚基 1 。这些毒素分泌到细胞外的环境，但他们的行为于真核细胞质内的目标。有些AB毒素旅游载体囊泡从细胞表面的内质网（ER），才进入细胞质2-4。在急诊室，不赞成从其余的毒素和移动通过一种蛋白质，在进行渠道的催化A链，以达到其胞质目标 5 。易位，胞质链是难以察觉，因为毒素贩运到急诊室是一个非常低效的过程：路由到最内在毒素降解的溶酶体，所以只有一小部分的表面结合的毒素达到高尔基体和ER 6 -12。

为了监视从ER在培养细胞的细胞质中的毒素易位，我们结合highl亚细胞分馏协议y的表面等离子体共振（SPR）13-15敏感的检测方法。毒素处理细胞的细胞膜选择性通透与毛地黄皂苷，使收集的一个细胞质部分，这是对涂有抗毒素A链抗体的SPR传感器随后灌注。抗体包传感器，可以捕捉和检测pg / mL的胞浆内毒素的数量。有了这个协议，它是可以遵循的毒素进入到细胞质的动力学特征易位事件的抑制作用。胞浆内毒素的浓度也可以从已知数量的A链已灌注在传感器的标准生成标准曲线计算。我们的方法代表了一种快速，敏感，定量检测系统不需要放射性标记或其他修改目标毒素。

Protocol

1。对毛地黄皂苷的制备离心管加入500μL100％的乙醇，并将其放置在在80℃10分钟的热块。 溶解在加热乙醇250μL2.5毫克毛地黄皂苷产生的毛地黄皂苷1％原液。 要产生0.04％毛地黄皂苷的工作液，添加40μL毛地黄皂苷原液HCN的缓冲液960μL（50 mM的HEPES pH值7.5，150 mM氯化钠，氯化钙2 2毫米，10毫米的N – ethylmaleimide，和一种蛋白酶抑制剂鸡尾酒）。 2。?…

Discussion

与现有方法的比较

我们基于SPR -易位检测的一种快速，敏感，定量的方法来检测毒素进入宿主细胞质交付。该技术并不需要放射性标记或其他修改的毒素，它可以应用到任何毒素，抗毒素A链抗体可。监测到细胞质的毒素通过现有的方法还依赖于一个分区成单独的细胞质和膜组分11，23，27，28的细胞提取物的亚细胞分馏协议。这些方法使用免疫印迹或radiolabels检测胞浆内?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国国立卫生研究院授予R01 AI073783光Teter。我们感谢援助，发展批判性阅读的手稿中的亚细胞分馏协议和海伦布瑞斯博士谢恩梅西。

Materials

Name of the reagent Company Catalogue number
Digitonin Sigma D141
Ethanol Acros 61509-0010
DMEM Invitrogen 11995065
Fetal Bovine Serum Atlanta Biologicals S11550
Ganglioside GM1 Sigma G7641
CTA Sigma C2398
PTS1 List 182
NHS (N-Hydroxysuccinimide) Pierce 24500
EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) Thermo Scientific 22981
Ethanolamine Sigma E0135
PBST Medicago 09-8903-100
Anti-CTA antibody Santa Cruz Biotech sc-80747
Anti-CTB antibody Calbiochem 227040
Anti-PTS1 antibody Santa Cruz Biotech sc-57639
Refractometer Reichert SR7000, SR7000DC
SPR sensor slides Reichert 13206060
Syringe pump Cole Palmer 780200C
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Tags

Toxin TranslocationSurface Plasmon ResonanceAB ToxinsCell-binding B SubunitVesicle CarriersEndoplasmic Reticulum (ER)Catalytic A ChainProtein-conducting ChannelCytosolic TargetToxin TraffickingLysosomesGolgi ApparatusSubcellular Fractionation ProtocolDigitonin PermeabilizationAnti-toxin A Chain AntibodyDetection Method
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Taylor, M., Banerjee, T., VanBennekom, N., Teter, K. Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance. J. Vis. Exp. (59), e3686, doi:10.3791/3686 (2012).
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