Name: mycobacterium tuberculosis extracellular vesicle enrichment through size exclusion chromatography
Uploaded: 2022-05-19T15:00:00
Description: Watch this Scientific Journal Video about Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography at JoVE.com

我们要感谢兽医学院和生物医学科学经验奖和大学研究委员会共享研究计划对NKG的支持，以及ATCC（奖项#2016-0550-0002）对KMD的资助。我们还要感谢安妮·辛普森的技术支持和BEI资源，NIAID，NIH提供以下试剂：单克隆抗结核分枝杆菌LpqH（基因Rv3763），IT-54（体外生产），NR-13792，单克隆抗结核分枝杆菌GROES（基因Rv3418c），克隆IT-3（SA-12）（体外生产），NR-49223和单克隆抗结核分枝杆菌LAM，克隆CS-35（体外产生），NR-13811。

科罗拉多州立大学机构生物安全委员会批准了本研究（19-046B）。 结核分枝杆菌 的培养和富含EV的培养物上清液的收获由训练有素的人员在高密闭实验室进行。在实施、确认并得到机构生物安全政策批准的有效灭活方法后，这些材料被移出高密闭区。在复制方案时，如果验证灭活或无菌过滤方法不可行，则需要在高密闭实验室中执行以下程序。
1. 粗山地车EV精矿的?...

<ol>
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结核分枝杆菌 细胞外囊泡是高度抗原的储库，这使它们成为开发诊断工具和未来疫苗的有吸引力的途径4，19，20。从历史上看，密度梯度超速离心已被用于将Mtb EV与其他可溶性分泌材料分离8。虽然该过程是有效的，但它也非常耗时，技术上具有挑战性，并且可能会影响所得EV准备的完整性...

病原体释放的细胞外囊泡（EV）可能是解锁控制传染病的新技术的关键1. 结核分枝杆菌（Mtb）是一种具有高度后果的病原体，每年感染约三分之一的世界人口，夺走数百万人的生命2。Mtb的EV生产有据可查，但在感染背景下，这些EV的生物发生和各种作用（即免疫刺激，免疫抑制，铁和营养获取）难以捉摸3，4，5。了解Mtb EVs组成的努力揭示了50-150nm脂质膜封闭的球体，这些脂质膜来自含有具有免疫学意义的脂质和蛋白质的质膜3，6。对Mtb EV在细菌生理学中的作用的研究揭示了细菌EV调节在响应环境压力对生存的重要性5。宿主- 病原体相互作用研究的解释更加复杂，但有证据表明，Mtb EV可以影响宿主的免疫反应，并可能作为有效的疫苗接种成分3，4，7。
到目前为止，大多数Mtb EV的研究都依赖于密度梯度超速离心进行囊泡富集8。这对小规模研究是有效的。然而，这种技术有几个技术和后勤方面的挑战。替代工作流程将多步离心与最终超速离心步骤相结合，以去除整个细胞和大碎片，以沉淀EV。这种方法的效率可能不同，并且通常导致可溶性非囊泡相关生物分子的低产量和共纯化，同时也影响囊泡完整性9。此外，此过程非常耗时，需要大量手动操作，并且由于设备限制，吞吐量非常有限。
本方案描述了密度梯度超速离心的替代技术：尺寸排阻色谱（SEC）。该方法已被证明适用于环境分枝杆菌，并且在当前的工作中，它已被外推到Mtb10。市售的色谱柱和自动馏分收集器可以提高制备的一致性，并减少对特定昂贵设备的需求。与密度梯度超速离心相比，也可以在很短的时间内完成该协议，从而提高通量。该技术在技术上的挑战性较小，使其更容易掌握，并且可以增加实验室间/实验室内的可重复性。最后，SEC具有高分离效率，并且温和，保持囊泡的完整性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20x MES SDS Running Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>NP0002</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>15705-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Fraction Collector</td>
			<td>IZON Science</td>
			<td>AFC-V1-USD</td>
			<td></td>
		</tr>
		<tr>
			<td>BenchMark Pre-stained Protein Ladder</td>
			<td>Invitrogen</td>
			<td>10748010</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra 6R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centricon Plus - 70 Centrifugal filter, 100 kDa cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC710008</td>
			<td>Ultrafiltration device used in step 1.1</td>
		</tr>
		<tr>
			<td>Electroblotting System</td>
			<td>ThermoFisher Scientific</td>
			<td>09-528-135</td>
			<td></td>
		</tr>
		<tr>
			<td>EM Grade Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar/Carbon 200 mesh Cu Grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>FCF200H-Cu-TA</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L (Alkaline Phosphatase), whole molecule, 1 mL</td>
			<td>AbCam</td>
			<td>ab6790</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>JEM-1400 Transmission Electron Microscope</td>
			<td>JOEL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BIOTEK</td>
			<td>Epoch</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Mycobacterium tuberculosis GroES (Gene Rv3814c)</td>
			<td>BEI Resources</td>
			<td>NR-49223</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Mycobacterium tuberculosis LpqH (Gene Rv3763)</td>
			<td>BEI Resources</td>
			<td>NR-13792</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Monocolonal Anti-Mycobacterium tuberculosis LAM, Clone CS-35</td>
			<td>BEI Resources</td>
			<td>NR-13811</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>NanoClean 1070</td>
			<td>Fischione Instruments</td>
			<td></td>
			<td>For plasma cleaning of the TEM grid</td>
		</tr>
		<tr>
			<td>Nanosight equipped with syringe pump and computer with NanoSight NTA software</td>
			<td>Malvern Panalytical</td>
			<td>NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane, Roll, 0.2 &#956;m</td>
			<td>BioRad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>ThermoFisher Scientific</td>
			<td>NP0323BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline, 1X without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BioRad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>qEV Original 35 nm 5/pk</td>
			<td>IZON Science</td>
			<td>SP5-USD</td>
			<td>SEC column</td>
		</tr>
		<tr>
			<td>SDS sample buffer</td>
			<td>Boster</td>
			<td>AR1112</td>
			<td>In-house recipe used in this procedure, however this product is equivalent</td>
		</tr>
		<tr>
			<td>SDS-PAGE gel chamber</td>
			<td>ThermoFisher Scientific</td>
			<td>EI0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmafast BCIP/NBT</td>
			<td>Millipore Sigma</td>
			<td>B5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain Plus Kit</td>
			<td>BioRad</td>
			<td>1610449</td>
			<td>In-house protocol used in this procedure, however this kit is equivalent</td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
	</tbody>
</table>

mycobacterium tuberculosis extracellular vesicle enrichment through size exclusion chromatography

细胞外囊泡（EV）在细菌感染中的作用已成为理解微生物生理学的新途径。具体而言， 结核分枝杆菌 （Mtb）EV在宿主 - 病原体相互作用和对环境压力的反应中发挥作用。Mtb EV也具有高度抗原性，并显示出作为疫苗成分的潜力。纯化Mtb EV的最常用方法是密度梯度超速离心。该过程有几个局限性，包括低通量，低产量，依赖昂贵的设备，技术挑战，并且可能对最终的制备产生负面影响。尺寸排阻色谱（SEC）是一种更温和的替代方法，可克服超速离心的许多局限性。该协议表明，SEC对MTB EV富集有效，并以快速和可扩展的方式生产高质量的MTB EV制剂，从而提高产量。此外，通过定量和鉴定程序与密度梯度超速离心的比较证明了SEC的优势。虽然EV数量（纳米颗粒跟踪分析），表型（透射电子显微镜）和含量（蛋白质印迹）的评估是为Mtb EV量身定制的，但提供的工作流程可以应用于其他分枝杆菌。

浓缩 来自结核分枝杆菌 （Mtb）的培养物滤液蛋白（CFP），定量，然后将3mg材料施用于尺寸排阻色谱（SEC）柱。蛋白质和颗粒浓度分别由支链氨基酸和NTA列举。蛋白质和颗粒回收的预期范围以及为这些结果获得的确切值报告在 表1中。远高于这些范围的值可能表示污染或色谱柱完整性问题。值明显较低，指向超滤步骤中的问题，因此需要将流过量与起始CFP和100R进行比较，以确定...

Watch this Scientific Journal Video about Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography at JoVE.com

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

该方案描述了尺寸排阻色谱，这是一种从培养物上清液中富集 结核分枝杆菌 细胞外囊泡的简单且可重复的技术。

结核分枝杆菌 通过尺寸排阻色谱实现细胞外囊泡富集

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. ...

该协议降低了从结核分枝杆菌，Mtb EVs富集细胞外囊泡所需的技术复杂性和时间。它为传统的超速离心和基于密度的技术提供了一种温和的替代方案。这种技术的主要优点是易于执行，使其更具可重复性。它提供了高产量的Mtb EV，具有最少的非EV蛋白共富集。演示该程序的将是Joanie Ryan，我们实验室的前研究生。首先，通过添加PBS的全部体积容量来制备100千道尔顿分子量截止离心过滤器。在4摄氏度下以2， 800倍G离心5分钟。丢弃流通物和任何剩余的PBS，并用Mtb CFP填充样品室至最大容量。在4摄氏度下以2， 800倍G离心，直到体积减小到超滤装置的最小体积。根据需要重复，直到整个样品充分减少。向含有浓缩物的过滤单元中添加 1X PBS，直至达到设备总容量。如前所述减少体积，重复此步骤五次，以确保完全洗涤和缓冲液更换。然后，根据超滤装置规格回收100千道尔顿浓缩CFP截留物或100R。用最小体积的1X PBS清洗过滤器至少三次，并用100R拉洗以最大限度地提高回收率。在PBS中使用几种稀释的样品，并用bicinchoninic测定法定量100R材料。一式三份测试样品。让体积排阻色谱或SEC色谱柱平衡至室温。使用塔式装置背面的电源开关打开AFC，如有必要，请按主菜单触摸屏上的设置来调整称重传感器的设置。在设置屏幕中，通过选择轮播，然后选择校准来对齐轮播。将转盘的 13 个小孔朝上插入 AFC 塔中，并通过按减号或加号按钮调整转盘，使流体喷嘴直接位于冲洗位置上方。从平衡的SEC色谱柱上取下盖子，然后将SEC色谱柱滑入相应的色谱柱安装座。小心地将其安装在AFC塔上。确保色谱柱上的射频识别标签面向AFC，并检查色谱柱与阀门之间的连接是否牢固。将废物出口管放入收集容器中。在设置屏幕中，选择收集计划，然后按减号或加号按钮将计数设置为 13，将大小设置为 0.5 毫升。将缓冲液体积设置保留为默认值 2.7 毫升，然后关闭收集计划窗口。从主屏幕中选择“开始收集”，然后选择“是”来确认收集参数。装入13个标记的1.7毫升微量离心管，盖子打开并指向转盘中心。通过选择“确定”推进AFC，然后安装色谱柱储液罐，然后按“确定”再次前进。按“是”（Yes）选择刷新色谱柱的选项，并添加一个0.2微米过滤PBS色谱柱体积以去除任何存储缓冲液。刷新完成后，按 OK 推进 AFC。将传送带盖放在 AFC 上，然后按 OK。使用移液器从色谱柱中去除多余的缓冲液。用1X PBS将3毫克100R样品带到500微升，并将样品添加到色谱柱顶部。制备一列体积的0.2微米过滤PBS。推进AFC并允许样品进入品格。样品完全进入色谱柱后，将先前制备的PBS添加到储液槽中。监控AFC的运行，同时它首先收集空隙体积，然后收集指定的分数。运行完成并且转盘返回到废物位置后，从转盘中取下盖子和馏分管。对于从 3 毫克 100R Mtb CFP 中收集的 0.5 毫米级分，使用微 BCA 以 1：3 稀释度测量蛋白质浓度，每个样品编号为 1 至 7 的馏分一式三份。对于编号为 5 至 13 的 0.5 毫升馏分，使用不稀释的 BCA 对每个样品一式三份。将SDS样品缓冲液添加到每个级分的10微升和5微克的CFP和100R中。在100摄氏度下煮五分钟。然后用分子量阶梯上样4至12%Bis-Tris凝胶，用于聚丙烯酰胺凝胶电泳或PAGE。使用 1X MES 电泳缓冲液和 200 伏电流运行 SDS-PAGE 凝胶 35 分钟。从凝胶盒中取出凝胶，并施加50伏电流至少一小时，将分离的蛋白质转移到0.2微米硝酸纤维素膜上。最后，进行蛋白质印迹分析以评估蛋白质。用银染色的SDS-PAGE凝胶显示了每个级分的总体蛋白质谱。数据显示，后面的馏分含有更多的蛋白质，类似于100R材料。检测LAM、LpqH和GroES的蛋白质印迹表明，蛋白质标志物在馏分中发生了变化。此处显示了固定前拉动的SEC级分1至3的透射电子显微镜或TEM图像。对Mtb EV的密度梯度超速离心进行负染色以进行TEM成像。此处显示了SEC级分1至3的纳米颗粒跟踪分析或NTA分布。采用纳米颗粒跟踪分析对密度梯度超速离心Mtb EVs进行了分析，并显示了尺寸分布。该协议已针对 3 毫克 100R 的 EV 纯化进行了优化。如果蛋白质上样量变化，则可能需要调整所得馏分的推荐稀释度和BCA策略。用这种技术制备的Mtb EV可用于下游成分和基于组学的研究。它们也适用于体外和体内实验。该技术为生产高质量的Mtb EV制剂提供了一种简单有效的方法，为未来Mtb EV实验的可重复性铺平了道路。

Research

JoVE Journal

Immunology and Infection

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

科研

免疫与感染

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

结核分枝杆菌 通过尺寸排阻色谱实现细胞外囊泡富集

结核分枝杆菌通过尺寸排阻色谱实现细胞外囊泡富集