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Summary
Abstract
Protocol
Results
Discussion
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Acknowledgements
Materials
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Immunology and Infection

朊病毒蛋白质错误折叠循环扩增

Published: November 07, 2012
doi:
10.3791/4075
, ,
1Department of Civil Engineering,University of Nebraska at Lincoln, 2Department of Medical Microbiology and Immunology,Creighton University

Summary

蛋白质错误折叠循环放大（PMCA）的的朊病毒转化和应变和物种障碍的研究是在体外试验。它也可以被用作朊病毒检测法。

Abstract

朊病毒是传染性病原体的动物和人类9,18导致的必然致命的传染性海绵状脑病（TSE）。的朊蛋白有两个不同的异构体，非传染性的主机的编码蛋 ​​白（PrP的C）和传染性蛋白（的PrP Sc）的，异常折叠的PrP〜C 8异构体。

朊病毒代理工作所面临的挑战之一是潜伏期长，临床症状主机接种后13前的发展。传统的授权和昂贵的动物生物测定研究。此外，生物化学和生物物理性能差的特点，其特殊的构象和聚集状态的PrP Sc的 。

的PrP sc可以转换的PrPÇ的PrP Sc的种子在体外 14。 PMCA是一种体外技术，需要阿德瓦ntage这种能力，利用超声和孵化周期产生大量的PrP Sc的 ，以更快的速度，从系统中含有过量的PrP C和微量的PrP Sc的种子19。这种技术已被证明，有效地重述物种和应变特异性的PrP Sc的转换，从蛋白Ç，干扰模拟朊病毒应变，并扩增的PrP Sc的非常低的水平，从受感染的组织，体液，及环境样品6,7,16， 23。

本文详细介绍了的PMCA协议，包括减少污染，产生一致的结果，这些结果和量化的建议。我们也讨论了几个的PMCA应用，包括株传染性朊病毒，朊病毒应变干扰，检测朊病毒的环境中产生和表征。

Protocol

1。准备设备使用Misonix 4000 3000或Misonix的超声波仪，（纽约法明达）连接到一个热电NESLAB EX-7水浴（纽因顿，NH），以保持恒定的温度为37°C。超声波清洗的样品在200微升薄壁的PCR管带与赛默飞世尔科技（马萨诸塞州沃尔瑟姆）获得的圆顶帽。 一个全新的超声发生器需要一个“磨合”期，连续工作9。甲两个月的磨合期由40秒的超声处理突发和孵育10分钟的周期与振幅电平设置?…

Representative Results

蛋白质错误折叠循环放大（PMCA）是用来放大的PrP Sc 在体外培养 7，12，14，19，24。一个成功的PrP Sc的扩增谱带强度的增加，如在图3中所示的PK抗朊病毒蛋白（19和30之间的迁移的kDa的仓鼠派生朊病毒株）的Western印迹上所示。后其谱带强度的增加PMCA表示扩增的PK-抗性的PrP Sc的材料。成功扩增仓鼠派生朊病毒蛋白，HaCWD和DY TME，在WB分析…

Discussion

放大传染性朊病毒蛋白质的挑战是在体内实验中的潜伏期长和费用。 PMCA技术是一种经济有效的手段，放大传染性朊病毒剂。几个实验室已证实的能力PMCA准确放大朊病毒株在体外 7，第9，第12，14，19,24。

朊病毒疾病可以物种之间发送。贝森和湿地，有效地接种仓鼠传染性貂脑病，这就产生了两个不同的地鼠源性朊病毒株5。在一个优雅的学…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢Vesper的铁玛丽·拉莫斯博士的批判性阅读的手稿。这项工作是由国家研究资源中心（P20 RR0115635-6，C06 RR17417-01和G20RR024001次）和美国国家神经疾病与中风研究所（2R01 NS052609）。

Materials

Reagent / Equipment Manufacturer Cat. Number
Misonix 3000 Misonix S-3000
Misonix 4000 Misonix S-4000
Tenbroeck Tissue Grinder Kontes 885000-0007
Neslab EX-7 Water Bath Thermo Electron Neslab EX-7
0.2 ml PCR Tube Strips Thermo Scientific AB-0451
Triton X-100 Sigma Aldrich T9284-100ML
Complete Protease Inhibitor Roche 11 697 498 001
EDTA J.T. Baker 4040-00
DPBS Mallinckrodt Baker Mediatech 21-031-CV
Versi-Dry Lab Soakers Fisher Scientific 14 206 28
Repti Therm Heater Zoo Med Laboratories, Inc. RH-4
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References

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Tags

Protein MisfoldingCyclic AmplificationPrionsTransmissible Spongiform EncephalopathyPrPCPrPScIncubation PeriodClinical SignsAnimal Bioassay StudiesBiochemical PropertiesBiophysical PropertiesIn Vitro TechniqueSonicationIncubation CyclesPrPSc SeedSpecies SpecificityStrain SpecificityPrion Strain InterferenceAmplificationFluidsEnvironmental SamplesPMCA ProtocolContamination ControlConsistent ResultsQuantification

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Saunders, S. E., Bartz, J. C., Shikiya, R. A. Protein Misfolding Cyclic Amplification of Prions. J. Vis. Exp. (69), e4075, doi:10.3791/4075 (2012).
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