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Introduction
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Imunologia e Infecção

实时颤振诱发的粪便中 CWD 朊的转化检测

Published: September 29, 2017
doi:
10.3791/56373
, , , , ,
1Dept. of Ecosystem and Public Health, Calgary Prion Research Units, Faculty of Veterinary Medicine,University of Calgary, 2Dept. of Molecular Biology,University of Wyoming, 3Canadian Food Inspection Agency,Lethbridge Laboratories

Summary

在这里, 我们提出一个协议来描述一个简单, 快速和有效的朊病毒放大技术, real-time 颤抖诱导转换 (RT-QuIC) 方法。

Abstract

RT QuIC 技术是一种敏感的体外无细胞朊病毒扩增法, 主要基于重组朊蛋白 (PrP) 基质的种子折叠和聚集, 以朊病毒种子为模板进行转化。RT QuIC 是一种新的高通量技术, 类似于 real-time 聚合酶链反应 (PCR)。淀粉样纤维生长的检测是基于染料 Thioflavin T, 这荧光在特定的相互作用与ᵦ片丰富的蛋白质。因此, 可以实时检测淀粉样蛋白的形成。我们试图建立一个可靠的非侵入性筛选试验, 以检测粪便提取物中的慢性 CWD 朊。在这里, 我们已经特别适应了 RT QuIC 技术, 以揭示 PrPSc播种活动的粪便 CWD 感染 cervids。最初, 我们准备的粪便提取物的播种活性在 RT-QuIC 中相对较低, 可能是由于粪便中的潜在化验抑制剂。为了提高粪便提取物的播种活性和去除电位测定抑制剂, 我们将粪便样品在含有洗涤剂和蛋白酶抑制剂的缓冲液中均匀化。我们还提交了样品到不同的方法, 以集中 PrPSc的基础上的蛋白质沉淀使用磷钨酸钠, 和离心力。最后, 通过优化的 RT QuIC 对粪便萃取物进行了测试, 其中包括在协议中对基体进行置换, 以提高检测的灵敏度。因此, 我们建立了一个 CWD 朊病毒播种活性的敏感检测方法, 在临床前 cervids 的粪便 QuIC, 这是一种实用的非侵入性 CWD 诊断的工具。

Introduction

朊病毒疾病或传染性海绵状脑 (东京) 是神经退行性疾病, 包括克雅氏病 (CJD) 在人类, 牛海绵状脑病 (疯牛病) 在奶牛, 病在绵羊和山羊, 和慢性浪费疾病 (CWD) 在 cervids 1,2。TSEs 的特点是有明显的海绵状的外观和神经元在大脑中的损失。根据 “仅蛋白质” 假说, 朊主要由 prpsc (“sc” 为病) 3, 错误型的主机编码的细胞朊蛋白, prpC。prpSc将 prpC转换为在ᵦ中丰富的构象4,5,6 , 它可以作为种子来绑定和转换其他 prpc分子。新生成的 PrPSc分子被合并成一个生长的聚合物7,8 , 它会分解成更小的寡聚物, 从而导致传染性核的数量增加。PrPSc易于聚合, 并部分抗蛋白酶9、10。

CWD 影响野生和养殖的麋鹿 (鹿黄花), 骡鹿 (Odocoileus 野驴), 白尾鹿 (WTD;Odocoileus 鹑)、驼鹿 (驼鹿驼鹿) 和驯鹿 (驯鹿 tarandus tarandus) 11,12,13。它被认为是最具传染性的朊病毒疾病与横向传播所青睐的 cervid 相互作用和环境持续性的传染性14,15。不同于其他朊病毒的疾病, PrPSc的积累和传染性被限制在大脑, 在 CWD 这些也发现在外围组织和体液, 如唾液, 尿液和粪便16,17,18。

免疫组织化学被认为是 CWD 诊断的金标准, 以检测 PrPSc分布和海绵状病变1920。ELISA 和更罕见的情况下, 西方印迹也用于 CWD 诊断。因此, 目前的朊病毒疾病诊断主要是基于检测朊在死后组织。CWD 的宰前诊断可通过扁桃体或直肠肛粘膜相关淋巴组织 (RAMALT) 活检获得;然而, 这个过程是侵入性的, 需要捕获的动物。因此, 使用容易接近的标本, 如尿和粪便, 将是一种切实可行的方法, CWD 朊病毒检测。然而, 那些排泄物港口相对地低浓度的朊低于目前诊断方法的检测限度。因此, 需要一个更敏感和高吞吐量的诊断工具。体外转换系统, 如蛋白质折叠循环放大试验 (PMCA) 21, 淀粉样播种试验, 和 real-time 颤抖诱导转换 (RT QuIC) 化验22,23,24是非常强大的工具, 可以利用 prpsc的蔓延能力模仿体外朊病毒转化过程, 从而放大少量 prp 的存在sc至可检测级别25 ,26。然而, RT-QuIC 方法利用了在β板二级结构中富集的转化产物可以具体地结合 thioflavin t (Th t) 的事实。因此, 重组 PrP (rPrP) 在种子转化后生长成淀粉样纤维, 并通过测量荧光的时间 t 表示为相对荧光单位 (RFU), 从而可以实时检测。一旦被监测, RFU 可用于评估相对播种活动, 以及定量参数, 如滞后阶段。滞后阶段表示到达阈值所需的时间 (h), 在此期间, 反应的早期阶段的 rPrP 转换低于 Th t 荧光的检测限度。明显滞后阶段的结束, 伴随着足够的淀粉样核的形成 (成核/伸长), 发生时, T 荧光超过阈值水平, 并成为积极的。淀粉样纤维的生长可以实时检测, 样品中含有的初始 PrPSc或播种活动通过分割产生更多的种子来放大。这些种子反过来诱发淀粉样纤维生长的快速指数阶段。

由于这种检测方法能够检测出 prpsc 24的低至 1 fg, 因此高灵敏度可以通过检测 prpsc在各种外围组织、排泄物或其他种类的标本, 窝藏低水平的传染性。RT-QuIC 肯定提供比其他化验的优势, 其重现性, 实用性, 快速 (少于50小时) 和低成本相比, 生物鉴定。避免了 PMCA 中使用的超声等技术复杂性;此外, 它是在胶带密封的微, 最大限度地减少了每井的气溶胶污染的风险。井格式可以在同一实验中对多达96样本进行分析。为了应对 rPrP 在体外转换分析中出现的误报和自发转换的复发问题, QuIC 中的阈值 (截止) 的实现非常有用。事实上, 根据负控制的结果 (负样本的平均 RFU +5 SD 27), 建立一个基线, 从中可以进行正负样本之间的区分。对每个样本使用四复制可以帮助定义一个样本为正数, 当至少50% 的复制显示一个正信号时,即跨越截断28。种子和基质之间的同源性在 RT-QuIC 中不需要, 如在以前的研究中, 仓鼠 rPrP 被发现是一个比较敏感的基板与人类 PrP 的同源基底相比,vCJD种子和绵羊病种子反应29. 仓鼠-绵羊嵌合 rPrP 也被认为是一个比人类 rPrP 更适合的基质, 以检测人类变体 CJD 朊30。因此, 在这种方法中使用不同种类的 rPrP 基质是非常普遍的。这种检测方法已成功应用于几种朊病毒疾病, 如零星 CJD 31,32,33, genetic 朊病毒疾病 34,疯牛病35,36,37, 病23,36, 和 CWD 38,39,40,41, 42。研究使用经加工的脑脊液, 全血, 唾液, 尿作为种子在 RT QuIC 都成功地检测 PrPSc 38,39,40,41,42. 为了培养可能含有淀粉样蛋白形成抑制剂的血浆样品的检测能力, Orrú et al。(2011) 通过梳理 PrPSc沉淀 (IP) 步骤和 RT QuIC (称为 “增强 QuIC”) 检测 (eQuIC), 制定了消除淀粉样蛋白形成的潜在抑制剂的策略。此外, 为了提高灵敏度, 在反应时间为 24 h 后采用了衬底置换步骤。最终, 低至 1 ag PrPSc可通过 eQuIC 30检测到。

为了净化粪便提取物, 清除粪便中可能的化验抑制剂, 从麋鹿在实验性口腔感染的前、临床分期采集的粪便样品均在含有洗涤剂和蛋白酶抑制剂的缓冲液中匀浆。粪便提取物进一步提交到不同的方法, 以集中 PrPSc在样品中利用蛋白质沉淀通过磷钨酸钠 (NaPTA) 沉淀。NaPTA 沉淀法, 首先由萨法尔et al.描述43, 用于将 PrPSc集中在测试示例中。NaPTA 与样品的孵化导致 prpSc而不是 prpC的优先沉淀。然而, 分子机制还不清楚。这一步骤还有助于遏制和防止 rPrP 的自发转化, 在某些情况下, 这种转换是被观察到的。最后, 以小鼠 rPrP (aa 23-231) 为基质, 对粪便萃取物进行了优化的 RT QuIC 测试, 并在协议中包括基片置换, 以提高检测的灵敏度。

研究结果表明, 这种改进的方法可以检测到极低浓度的 CWD 朊, 增加了粪便样品中的检测和特异性的灵敏度, 而不需要 NaPTA 沉淀和基质置换。这种方法可能适用于其他组织和体液, 可以很好地用于野生和圈养 cervids 的 CWD 监测。

Protocol

1. 使用粪便材料的 RT QuIC cervid 粪便提取物的制备 使粪便匀浆增加1克粪便物质到10毫升的粪便萃取缓冲液 (20 毫米磷酸钠, pH 7.1, 130 毫米氯化钠,0.05% 吐温 20, 1 毫米 PMSF 和1x 完整蛋白酶抑制剂, 无 EDTA), 以最后浓度为 10% (w/v)。均匀化缓冲器可在使用前准备, 并存储在-20 和 #176; C. 质粪丸 (1 克) 和缓冲 (10 毫升) 在管 (例如, gentleMACS M 管) 使用 dissociator 与预先设定…

Representative Results

10% (CWD) 制备的粪提取物能够 QuIC 反应, 但检测灵敏度较低, 为27。使用一个特定的缓冲区, 粪便均匀化是一个关键步骤, 以避免高背景荧光在 RT-QuIC 反应与使用鼠标 rPrP 衬底, 而不是鹿 rPrP, 允许获得更具体的结果27。NaPTA 降水的增加减少了 rPrP 在 RT-QuIC (图 1) 中的自发转换, 而不抑制反应的播种活动的放大 (<strong class="…

Discussion

QuIC 以前被用来检测 CWD 朊的尿液和粪便提取物的口腔感染白尾鹿和骡鹿38。本手稿中所示的系统是一种适应的 RT-QuIC 法。另外的步骤被纳入 “经典” RT QuIC 测定, 以提高检测和灵敏度的 CWD 朊在粪便材料的感染动物。

在粪便提取物中, 检测灵敏度低, 使我们对 QuIC 协议进行了改进。为了实现敏感的体外检测粪便中的朊, 为了去除干扰朊病毒转化和/或传播的…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢 Dr. Caughey (NIH 岩石山实验室) 提供培训和 cervid PrP 细菌表达质粒。SG 得到加拿大研究主席计划的支持。我们承认这项研究的资金来自加拿大基因组、艾伯塔朊病毒研究所和艾伯塔省农业和林业, 通过基因组艾伯塔省和卡尔加里大学支持这项工作。我们承认来自玛格丽特的动物研究基金会的研究补助金。

Materials

Materials
Acrodisc seringe filters PALL 4652
amicon Ultra-15 Centrifugal filter Unit Millipore UCF901024
BD 10 ml seringe VWR CA75846-842
Chloramphenicol Sigma-Aldrich C0378
Corning bottle-top vacuum filters Sigma-Aldrich 431118
Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich E4884
gentleMACS M Tube Miltenyi Biotec 130-093-236
Guanidine hydrochloride Sigma-Aldrich G4505
Imidazole Sigma-Aldrich I5513
Isopropanol Sigma-Aldrich I9516
Kanamycin sulfate Sigma-Aldrich 60615
Luria-Bertani (LB) broth ThermoFisher Scientific 12780029
Magnesium chloride Sigma-Aldrich M9272
N2 supplement (100X) ThermoFisher Scientific 15502048
N-lauroylsarcosine sodium salt (sarkosyl) Sigma-Aldrich ML9150
Nanosep centrifugal devices with omega membrane 100K PALL OD100C34
Nunc sealing tapes ThermoFisher Scientific 232702
Parafilm M VWR 52858-000
phenylmethylsulfonyl fluoride (PMSF) Sigma-Aldrich P7626
Protease inhibitor tablet Roche 4693159001
Sodium chloride Sigma-Aldrich S3014
Sodium deoxycholate Sigma-Aldrich D6750
Sodium dodecyl sulfate (SDS) Calbiochem 7910-OP
sodium phosphate Sigma-Aldrich 342483
Sodium phosphate dibasic anhydrous Sigma-Aldrich S9763
Sodium phosphate monobasic monohydrate Sigma-Aldrich S9638
Sodium phosphotungstate hydrate (NaPTA) Sigma-Aldrich 496626
Thioflavin T Sigma-Aldrich T3516
Tris-Hydroxy-Methyl-Amino-Methan (Tris) Sigma-Aldrich T6066
Triton-100 Calbiochem 9410-OP
Tween 20 Sigma-Aldrich P7949
Name Company Catalog Number Comments
Commercial buffers and solutions
BugBuster Master Mix Nogagen 71456-4
Ni-NTA superflow Qiagen 1018401
Phosphate-buffered saline (PBS) pH 7.4 (1X) Life Technoligies P5493
UltraPure Distilled Water Invitrogen 10977015
Name Company Catalog Number Comments
Standards and commercial kits
Express Autoinduction System 1 Novagen 71300-4
Pierce BCA Protein Assay Kit ThermoFisher Scientific 23227
Name Company Catalog Number Comments
Equipment setup
AKTA protein purification systems FPLC GE Healthcare Life Sciences
Beckman Avanti J-25 Centrifuge Beckman Coulter
Beckman rotor JA-25.50 Beckman Coulter
Beckman rotor JA-10 Beckman Coulter
FLUOstar Omega microplate reader BMG Labtech
gentleMACS Dissociator Miltenyi Biotec 130-093-235
Name Company Catalog Number Comments
Sofware
MARS Data Analysis BMG Labtech
GraphPad Prism6 GraphPad software
DOWNLOAD MATERIALS LIST

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Tags

CWD PrionsFecal MaterialReal-time Quaking-induced Conversion AssayCervidChronic Wasting DiseaseSensitive DetectionHigh ThroughputSodium Phosphotungstic Acid PrecipitationRT-QuIC AssayThioflavin T

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Cheng, Y. C., Hannaoui, S., John, T. R., Dudas, S., Czub, S., Gilch, S. Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material. J. Vis. Exp. (127), e56373, doi:10.3791/56373 (2017).
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