Name: protein misfolding cyclic amplification of prions
Uploaded: 2012-11-07T12:00:00
Description: Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

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）。

1。准备设备<ol><li>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。 </li><li>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?...

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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...

<table border="1"><tbody><tr><td>试剂/设备</td><td>生产厂家</td><td>猫。数</td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck组织研磨机</td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> NESLAB EX-7水浴</td><td> Thermo Electron公司</td><td> NESLAB EX-7 </td></tr><tr><td> 0.2毫升PCR管条</td><td> Thermo Scientific的</td><td> AB-0451 </td></tr><tr><td>的Triton X-100的</td><td> Sigma Aldrich公司</td><td> T9284-100ML </td></tr><tr><td>完整的蛋白酶抑制剂</td><td>罗氏公司</td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT贝克</td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt公司贝克敏达</td><td> 21-031-CV </td></tr><tr><td>砂劳越干实验室Soakers </td><td> Fisher Scientific则</td><td> 14 206 28 </td></tr><tr><td> Repti千卡加热器</td><td>动物园地中海Laboratories公司</td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

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。

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...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

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。

朊病毒蛋白质错误折叠循环扩增

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Research

JoVE Journal

Immunology and Infection

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

科研

免疫与感染

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

实时颤振诱发的粪便中 CWD 朊的转化检测

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

重组呼吸道合胞病毒的生成、扩增和滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

基于 TurboID 的接近标签，用于蛋白质-蛋白质相互作用网络的植物中识别

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...