Name: assays for studying the role of vitronectin in bacterial adhesion and serum resistance
Uploaded: 2018-10-16T12:30:00
Description: Watch this Scientific Journal Video about Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance at JoVE.com

这项工作得到了来自安娜和埃德温-伯杰基金会、Hierta、气流和 Edla 约翰逊基金会、瑞典医学研究理事会 (赠款号 K2015-57X-03163-43-4、www.vr.se)、大学癌症基金会的赠款的支持。在马尔默的医院, 地形协会 (Forssman 基金会), 和斯科讷郡议会的研究和发展基金会。

1. Vn 作为细菌表面蛋白配体的分析
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应用流式细胞仪检测细菌表面的 Vn 结合 注: 在流式细胞术中, 我们使用侧散射和正向散射来门正事件。为了检查与 Vn 的相互作用, Hif 临床分离 (n=10)7与大肠杆菌BL21 (DE3) 一起选择作为阴性对照 (图 1a)。<ol>
<li>培养 Hif 在脑心脏输液 (BHI) 培养基中的临床分离剂, 辅以10µg/毫升 NAD 和氯...

<ol>
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细菌病原体将 Vn 招募到细胞表面, 并利用此补充调节器防止补体因子的沉积和膜攻击复杂2的完成。Vn 还充当细菌表面蛋白和宿主细胞表面受体之间的桥梁分子, 从而使病原体能够附着在上皮细胞表面并随后调解内部化。在本研究中, 我们描述了可用于估计 i 的协议: vn 与细菌细胞表面的结合, ii) 蛋白质-蛋白质相互作用和亲和常数, iii. vn 在提供血清耐药性和增强的作用附着在宿主?...

Vitronectin (Vn) 是一种重要的人类糖蛋白, 通过调节纤溶系统来维持稳态。在 C5b6-7 复合形成和 C9 聚合过程中, Vn 还通过抑制末端补道来发挥补充调节作用。一些细菌病原体已被证明要招募 Vn 到细胞表面作为抵抗补体沉积1,2,3的手段。此外, Vn 在细菌和宿主上皮细胞受体之间充当 "三明治" 分子, 从而促进病原体的附着和内部化2,4,5。Vn 与细菌细胞表面的结合是由其他目前未识别的蛋白质介导的。因此, 充分阐明 vn 结合在规避软管免疫反应方面的功能作用, 将需要鉴定 vn 的蛋白质。
确定 vn 结合蛋白的最初步骤是测试是否有兴趣的病原体可以结合纯化的 vn. 流式细胞术是一种简便而直接的方法, 用于确定 vn 是否与病原细胞结合。在本研究中, 我们评估了 Vn 与不同类型的流感嗜血杆菌 (Hif) 临床分离6的结合。本文描述的方法是定量的, 可用于区分各种细菌菌株的结合能力。在以前的研究中, 我们将 Hif 的蛋白质 H (PH 值) 描述为 Vn 结合蛋白7。因此, 在本研究中, 用所述的方法比较了野生型 (WT) Hif 和 Hif M10Δlph突变体的 Vn 结合电位。
一旦确定病原体结合 Vn, 第二步是表征表面蛋白质组, 以确定潜在的 Vn 结合蛋白。可以使用多种方法来实现8、9, 但本报告没有介绍这些方法。最适合检测蛋白质-蛋白质相互作用的方法是在大肠杆菌中制备表达选择的细菌表面蛋白, 并通过亲和层析纯化。在这里, 我们使用了 PH 及其分子相互作用与 Vn 来说明该方法。重组 PH 和 Vn 之间的相互作用采用酶联免疫吸附试验 (ELISA)7和最近开发的无标签技术 (称为生物层干涉法 (BLI)10,11) 进行了表征。虽然 ELISAs 可用于确认蛋白质-蛋白质相互作用, 但 BLI 提供有关相互作用的动力学参数的详细数据。
为了研究 Vn 在细菌粘附中的功能作用, 可以使用两种不同的检测法。这里描述的第一种检测方法是直接测量对 Vn 涂层玻璃表面的细菌附着, 而第二种检测则检查附着在上皮细胞表面。对于第一种检测方法, 玻璃玻片涂有 Vn, 用革兰染色和显微显微镜评估了 WT 或突变 Hif 菌株的结合度。这种技术可以根据结合 Vn12的能力, 轻松区分细菌。然后, 通过在 II. 型肺泡上皮细胞的单层添加培养细菌, 对哺乳动物细胞的细菌黏附性进行分析;通过计数菌落形成单位 (CFUs) 的数量来评估细菌附着。附着和内化细菌可在 Vn4、13的存在或缺失时加以区分。
利用血清杀灭试验 (即血清杀菌活性) 评价了 Vn 在细菌性血清耐药性中的作用。为了评价 vn 在血清耐药性中的意义, 与正常人血清 (NHS) 的杀菌活性进行了比较。该方法可以很容易地区分 Vn 结合与非结合细菌的血清耐药性。我们用这种方法研究了 Vn 在几种细菌病原体的耐药性中的作用4,12。
许多方法已经报告了研究寄主病原体的相互作用。在这里, 我们描述了一套协议, 可以很容易地适应任何病原体的研究, 以评估 Vn 在发病机制中的作用。我们使用各种病原体测试了这些协议, 并选择了 Hif 作为本报告的示例。

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			<td height="21" style="height:21px;width:335px;">1.5 mL thermomixter</td>
			<td style="width:288px;">Eppendorf</td>
			<td style="width:119px;">5355</td>
			<td style="width:272px;">dry block heating and cooling shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL polystyrene round-bottom tube&#160;</td>
			<td>BD Falcon</td>
			<td>60819-138</td>
			<td>12 &#215; 75 mm style</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">5% CO2 supplied incubator&#160;</td>
			<td style="width:288px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">BBD6220</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">6 mL polystyrene round-bottom tube&#160;</td>
			<td style="width:288px;">VWR</td>
			<td>89000-478</td>
			<td>12 &#215; 75 mm style with cap</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">24-well plates</td>
			<td style="width:288px;">BD Falcon</td>
			<td>08-772-1H</td>
			<td style="width:272px;">Cell culture grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">30% Hydrogen peroxide (H2O2) solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">H1009-100ML</td>
			<td>Laboratory analysis grade</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">75 cm2 tissue culture flask</td>
			<td style="width:288px;">BD Falcon</td>
			<td>BD353136</td>
			<td>Vented</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well black flat bottom plate</td>
			<td style="width:288px;">Greiner Bio-One</td>
			<td>655090</td>
			<td>Tissue culture treated &#181;Clear black plates</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">A549 Cell Line human</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">86012804-1VL</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Cell detachment enzyme (Accutase)&#160;</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>A6964-500ML</td>
			<td style="width:272px;">Cell Culture Grade</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:335px;">AR2G sensors</td>
			<td style="width:288px;">Pall Life Science</td>
			<td>18-5095</td>
			<td style="width:272px;">Sensor to immobilized protein by amino coupling&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Acetone</td>
			<td style="width:288px;">VWR</td>
			<td>97064-786</td>
			<td>Analysis grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Bovine Serum Albumins (BSA)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>A2058</td>
			<td style="width:272px;">Suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bibulous paper&#160;</td>
			<td style="width:288px;">VWR</td>
			<td>28511-007</td>
			<td></td>
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			<td height="43" style="height:43px;width:335px;">Bio-layer interferometer</td>
			<td style="width:288px;">Pall Life Science</td>
			<td style="width:119px;">FB-50258</td>
			<td style="width:272px;">Bilayer interferometry measuring equipment</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Crystal violet solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>HT90132-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">C4BP (C4b binding protein)</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td style="width:119px;">A109</td>
			<td>Bought as Frozen liquid form</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">Calcium chloride (CaCl2)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>C5670-500G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Carbol-fuchsin solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>HT8018-250ML</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Citric acid</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>251275-500G</td>
			<td>American Chemical Society (ACS) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Decolorizing solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>75482-250ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">E. coli host (E. Coli BL21)</td>
			<td style="width:288px;">Novagen</td>
			<td>69450-3</td>
			<td>Protein expression host</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">F12 medium</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>D6421</td>
			<td style="width:272px;">Cell Culture Grade</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:335px;">Flow cytometer&#160;</td>
			<td style="width:288px;">BD Biosciences</td>
			<td style="width:119px;">651154</td>
			<td style="width:272px;">Cell analysis grade for research applications&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Fetal Calf Serum (FCS)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>12003C</td>
			<td style="width:272px;">Suitable for cell culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Normal human serum (NHS)</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td>NHS</td>
			<td style="width:272px;">Pooled human serum</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">FITC-conjugated donkey anti-sheep antibodies&#160;</td>
			<td style="width:288px;">AbD Serotec</td>
			<td>STAR88F</td>
			<td style="width:272px;">Polyclonal</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Gentamicin</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>G1397</td>
			<td style="width:272px;">Cell culture grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glucose</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Gelatin</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>G9391</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:288px;">Marienfeld</td>
			<td>640210</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">HRP-conjugated anti-His tag antibodies</td>
			<td style="width:288px;">Abcam</td>
			<td style="width:119px;">ab1269</td>
			<td style="width:272px;">Polyclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Human factor H</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td style="width:119px;">A137</td>
			<td>Bought as Frozen liquid form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">C4BP</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td style="width:119px;">A109</td>
			<td>Frozen solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Human serum albumin</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1653-10G</td>
			<td>lyophilized powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histidine affinity resin column (HisTrap HP)</td>
			<td style="width:288px;">GE Health Care Life Science</td>
			<td style="width:119px;">17-5247-01</td>
			<td>Columns prepacked with Ni Sepharose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">His-tagged PH</td>
			<td style="width:288px;"></td>
			<td style="width:119px;"></td>
			<td>Recombinantly expressed and purified in our lab</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Iodine solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>HT902-8FOZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Methanol</td>
			<td style="width:288px;">VWR</td>
			<td>BDH1135-1LP</td>
			<td>Analysis grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">&#160;Microscope</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:119px;">IX73</td>
			<td style="width:272px;">Inverted microscope</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Microscope slides</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>S8902</td>
			<td style="width:272px;">plain, size 25 mm &#215; 75 mm&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">Magnesium chloride (MgCl2)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>M8266-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid containg C terminal 6x His-tag on the backbone (pET26(b))</td>
			<td style="width:288px;">Novagen</td>
			<td>69862-3</td>
			<td>DNA vector</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Polysorb microtitre plates&#160;</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>M9410</td>
			<td style="width:272px;">For ELISA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Potassium hydroxide (KOH)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">6009</td>
			<td>American Chemical Society (ACS) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Sheep anti-human Vn antibodies</td>
			<td style="width:288px;">AbD Serotec</td>
			<td>AHP396</td>
			<td style="width:272px;">Polyclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Shaker&#160;</td>
			<td style="width:288px;">Stuart Scientific</td>
			<td style="width:119px;">STR6</td>
			<td>Platform shaker</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">Tissue culture flask</td>
			<td style="width:288px;">BD Falcon</td>
			<td style="width:119px;">3175167</td>
			<td style="width:272px;">75 cm2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">&#160;Thermomixer&#160;</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>T3317</td>
			<td>Dry block heating and cooling shaker</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Tetramethylbenzidine</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>860336-100MG</td>
			<td>ELISA grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Vitronectin (Vn) from human plasma</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>V8379-50UG</td>
			<td>cell culture grade</td>
		</tr>
	</tbody>
</table>

assays for studying the role of vitronectin in bacterial adhesion and serum resistance

细菌利用补充调节器作为逃避宿主免疫反应的手段。在这里, 我们描述了评估在细菌细胞表面 vitronectin 获取的作用的协议, 以抵抗宿主免疫系统。流式细胞术实验确定人血浆 vitronectin 作为一种配体的细菌受体外膜蛋白 H 型流感嗜血杆菌 f。采用酶联免疫吸附法对纯化的重组蛋白 H 与 vitronectin 蛋白相互作用进行了表征, 并利用生物层干涉方法评估了结合亲和性。使用正常和 vitronectin 耗尽的人血清进行血清耐药性测定, 证实了 vitronectin 与蛋白 H 在细菌细胞表面的结合对宿主免疫应答的生物学重要性。采用无 vitronectin 涂层的玻片和革兰染色, 分析了 vitronectin 在细菌粘附中的重要性。最后, 对人肺泡上皮细胞膜的细菌黏附性进行了研究。这里描述的协议可以很容易地适应对任何细菌物种感兴趣的研究。

用流式细胞仪测定了对细菌表面的 Vn 结合。在这项研究中, 所有的 Hif 临床分离试验都是在细胞表面进行的。大肠杆菌阴性对照菌株未观察到 Vn 与细胞表面的相互作用 (图 1A)。如图 1B所示, PH 值是 Hif 细胞表面的一种主要的 Vn 结合蛋白。Hif 应变 M10 对 vn 的束缚导致了种群的转移, 而 Hif M10Δlph</...

Watch this Scientific Journal Video about Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance at JoVE.com

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

本报告描述了用于表征细菌外膜蛋白与人体补体调节器 vitronectin 之间相互作用的协议。该协议可用于研究 vitronectin 在任何细菌物种中的结合反应和生物学功能。

Vitronectin 在细菌黏附和血清耐药性中作用的实验研究

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

这种方法可以帮助回答宿主-病原体相互作用领域的关键问题，例如细菌病原体如何利用人类蛋白质在人类中引起疾病。这种技术的主要优点是它可以回答是否有任何病原体可以利用维特龙素建立宿主的感染。我们将重点介绍 Vitronectin，一种参与细胞外基质的宿主蛋白，但也作为补充激活决定途径的补充抑制剂。这些事实对于引起人类疾病的病原体非常重要和极具吸引力。为准备流动细胞学，用含有250纳米摩尔维特龙素的50微升阻断缓冲液重新填充细菌颗粒。然后，在室温下孵育样品一小时，不摇晃。孵育后，在3，500g的离心下对细菌进行5分钟的造粒。然后，使用 PBS 和类似的离心步骤清洗颗粒三次。洗涤后，在PBS BSA中，在1至100稀释时，在细菌颗粒中加入50微升原发性绵羊抗人维特龙素多克隆抗体。在室温下孵育悬浮液一小时。然后，用PBS清洗细菌三次，以去除未结合的抗体。接下来，加入50微升PBS BSA含有荧光素同位素的驴抗羊多克隆抗体。在黑暗中在室温下孵育一小时。最后，经过三个洗涤步骤，用300微升PBS重新暂停细菌颗粒，并通过流式细胞仪进行分析。为了准备ELISA，将100微升的蛋白质溶液分配到聚索普微刺激板的每个孔中。用盖子合上板，在四摄氏度下储存过夜，以方便蛋白质固定在微刺激板井上。通过倒置在水槽上，丢弃微刺激板中的溶液。然后，用每口300微升PBS洗井三次。在室温下用 PBS 将涂层孔阻塞一小时，其重量体积为 2.5 百分比 BSA。去除阻塞溶液后，用每口300微升PBST洗井三次。现在，在每个样品中加入100个50纳米摩尔重组的微升，将他标记的PH。在控制井中，仅添加 100 微升 PBS BSA。在室温下孵育板一小时。孵育后，丢弃蛋白质溶液，用每井300微升PBST洗涤三次，去除未绑定的蛋白质。然后，加入100微升PBS BSA含有马萝卜过氧化物酶结合抗他的多克隆抗体。在室温下孵育一小时后，用每井300微升PBST洗井三次。通过向每一井添加 100 微升 ELISA 检测试剂来检测抗原-抗体复合物。然后，每井加入50微升一个摩尔硫酸，以停止反应。使用微孔板读卡器测量 450 纳米的孔的光学密度。根据制造商的指南，使用胺耦合方法在胺反应传感器上固定人 Vitronectin。使用 PBS，连续稀释重组 PH 配体从零到四微摩尔，并将生成的溶液转移到 96 孔黑色平底微蒂板。使用生物层干涉测量仪器在30摄氏度下运行实验。PBS 中每毫升 Vitronectin 溶液的 10 微升，作为一滴，放在玻璃显微镜幻灯片上。在室温下，让落差在幻灯片上干燥 30 分钟。将蛋白质涂层的玻璃幻灯片浸入含有 PBS 的烧杯中浸两秒钟，以去除多余的未涂覆蛋白质，将其洗涤三次。现在，将 10 毫升的新鲜 Hif 培养剂加入无菌塑料培养皿中，并将涂层玻璃幻灯片淹没在培养基中。在37摄氏度下孵育一小时，在20 RPM时摇晃。孵育后，将幻灯片三次淹没在装满PBS的烧杯中，然后像文本协议中描述的语法染色使受约束细菌目对目，以去除任何未绑定的细菌。培养 A549 上皮细胞，如文本协议中所述。在37摄氏度下对细胞进行一次洗涤和三辛化后，使用每毫升根霉素含有5微克的完整介质将细胞悬浮液稀释至5000个细胞。在细菌感染之前，用F12介质代替井中的介质，并在37摄氏度下孵育过夜。在室温下用一毫升PBS清洗细胞单层三次。然后，将板放在冰和200微升预冷F12介质上，每毫升维特罗内丁含有10微克。在四摄氏度下孵育一小时后，通过移液丢弃溶液，并在室温下用一毫升PBS清洗细胞层两次。在每个井中加入100个新鲜生长的Hif野生型的微升。在37摄氏度下孵育板两小时。然后，用移液器吸气介质，用PBS清洗 A549 上皮细胞三次。每井的细胞分离溶液添加50微升，并在37摄氏度下进行5分钟的孵育。接下来，每井添加50微升F12完整介质，以停止酶反应。将上皮细胞从每一个井转移到一个六毫升的玻璃管，里面装着四个玻璃珠。通过涡旋两分钟，在室温下使细胞保持。稀释细胞 100 倍后，将稀释样品的 10 微升板盘放到巧克力加糖板上。在37摄氏度下过夜孵育后，计算菌落。为了分析维特龙素依赖性对人体血清杀菌活性的耐药性，首先培养和颗粒菌，如文本协议所述。离心后，用一卷葡萄糖-胶质-维龙缓冲液重新填充细菌颗粒。现在，在 100 微升 DGVB 加加含有 5% 血清的 100 微升中加入 1，500 CFU 细菌。在37摄氏度下孵育样品15分钟，在300 RPM下摇动。在零分钟和15分钟内从反应混合物中去除10微升等分。将等分放在巧克力加糖上，在37摄氏度下孵育。在巧克力加糖板孵育后，计算板上出现的菌落，并计算文本协议中描述的细菌的百分比。本研究中测试的所有Hif临床分离物都将维特龙素招募到细胞表面，由流式细胞学测定。野生型Hif菌株对维特龙素的结合导致种群的转移，而突变体没有结合维特龙素。ELISA估计了主要维特龙素结合蛋白PH和维特龙素之间的蛋白质-蛋白质相互作用。结果表明PH与维特龙素和正控相对于负对的显著相互作用。使用生物层干涉测量技术实时估计PH和维特龙素之间的相互作用，具有2.2微摩尔的亲和力。此外，与野生型细胞相比，细胞表面缺乏PH表达的细菌减少了对维特龙素涂层玻璃表面的依从性。此外，上皮细胞表面的维特龙素的存在显著增强了Hif的粘附性。为了估计维特龙素的补性抑制活性，对血清中位杀戮进行了检查。野生型Hif细胞表现出比突变细胞更高的血清耐药性。没有细菌在热灭活血清的存在下死亡。有趣的是，野生型Hif在维特罗内辛贫化血清中表现出的存活率降低，而补充维特罗内辛增加了细菌存活率。然而，突变菌株没有对维特龙素的耗竭作出反应，因为它不能将维特龙素招募到细胞表面。一旦掌握，这种技术可以在三到四天内完成任何病原体，并记住使用细菌病原体的新鲜培养。看完这段视频后，您应该对如何分析与病原体结合的 Vitronectin 有很好的了解。除了这种技术，可以使用蛋白质组学和西印迹来识别细菌的维特龙素结合表面蛋白。该技术对于宿主-病原体相互作用领域的研究人员以及探讨细菌毒性机制十分重要。

Research

JoVE Journal

Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

科研

免疫与感染

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

基于聚丙烯酰胺的多井格式实验研究细胞外基质硬度对黏附细胞细菌感染的影响

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...