Name: protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection
Uploaded: 2011-05-02T15:00:00
Duration: 12 min 9 s
Description: Watch this Scientific Journal Video about Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection at JoVE.com

支持这项研究是由美国国立卫生研究院（NIH）的美国（RO1 AI68002）。

1。重组SARS - CoV的RBD的蛋白制备<ol><li>准备磷酸钙转染试剂<ol><li> 2X哈佛商学院的缓冲液配制：16克氯化钠，0.4克的Na 2 HPO 4 7H 2 O，13.0克HEPES混合在一起。调整pH值至7.00，并带来总量1000毫升蒸馏水中。经过过滤消毒，分装解决方案，并储存在-20 ° C 提示：任何pH值的变化会影响转染结果。因此，最好是来测试几个pH值约7.00（例如，6.99，7.00，7.01），并找到最好使用下面介绍的方法转染。 </li><li> 2.5米氯化钙2准备：在终体积为200毫升添加73.5克的氯化钙2 · 2H 2 O蒸馏水。过滤器的解决方案，并储存在-20 ° C </li></ol></li><li>重组质粒转染和蛋白的纯化<ol><li>分割前汇合24 h转染293T细胞在50-70％。成长中的T - 175厘米2组织在40毫升的DMEM含10％热灭活胎牛血清和1％青霉素/链霉素（P / S）在37 ° 5％的CO 2（HI）的彗星的培养瓶中的细胞。 </li><li>应使所有的转染试剂室温染前。这些试剂包括哈佛商学院的2倍，2.5M 氯化钙 ，DIH 2 O，和rRBD质粒6。 提示：最后的重组质粒构建用于蛋白表达，应该包含一个信号肽，以确保培养上清分泌表达的重组蛋白。可能会增加6倍His标签易于纯化的表达蛋白C -末端。 </li><li>准备之一50毫升（A）和一个15毫升（b）屋宇署猎鹰管，并加试剂如表1所示的管。 A.管B管加入2X哈佛商学院的缓冲区，加2.5M 氯化钙和rRBD质粒，并带来DIH 2 O量的要求 <table border="1"><tbody><tr><td colspan="4"> A.一个T - 175厘米2组织培养瓶（4000μL/烧瓶）：准备rRBD表达 </td></tr><tr><td colspan="2"> 管中 </td><td colspan="2"> 管B </td></tr><tr><td> 2X哈佛商学院</td><td> 2000微升</td><td> 2.5M氯化钙2 </td><td> 200微升</td></tr><tr><td></td><td></td><td> rRBD质粒DNA </td><td> 40微克</td></tr><tr><td></td><td></td><td> DIH 2 O来 </td><td> 2000微升 </td></tr><tr><td colspan="4"> B.一个100毫米的培养皿（1000μL/皿）：SARS pseudovirus生产准备</td></tr><tr><td colspan="2"> 管中 </td><td colspan="2"> 管B </td></tr><tr><td> 2X哈佛商学院</td><td> 500μL， </td><td> 2.5M氯化钙2 </td><td> 50微升</td></tr><tr><td></td><td></td><td> SARS - CoV的S质粒DNA </td><td> 5微克</td></tr><tr><td></td><td></td><td> HIV - 1质粒（pNL4 3.luc.RE） </td><td> 5微克</td></tr><tr><td></td><td></td><td> DIH 2 O来 </td><td> 500μL， </td></tr></tbody></table> 表1转染混合物制备和卷 在表1中列出的卷是一个转单位。如果更多的烧瓶或菜肴用于转染，卷相应的调整。 </li><li>添加到管中滴加方式一管B的DNA钙的解决方案，同时保持常数和温柔的一个旋涡混合。让混合物在室温下坐20-30分钟。磷酸钙转染成功的关键取决于形成的沉淀物的大小和形状。因此，应混合振荡不断，慢慢地满足这一要求，因此，提高转染疗效。 </li><li>在滴加甚至添加到293T细胞（4000微升/瓶）的混合物。培养细胞在37 ° C在5％CO2培养箱中培养。 </li><li>更换新鲜的无血清的OPTI - MEM我降低血清中（50毫升/瓶）8-10 h后转染培养液中。两个多天在相同条件下，继续培养细胞。 </li><li>收集上清含有表示rRBD蛋白72 h后转染。在15分钟6000转离心去除细胞碎片。添加蛋白酶抑制剂的鸡尾酒收集上清和储存在4 ° C过夜。 </li><li>第二天，从培养上清净化rRBD重组蛋白，用镍NTA Superflow以下制造商的指示。 </li><li>使用Amicon超-15浓度管浓缩纯化蛋白质。蛋白浓度后，添加PBS的浓度管和离心机再次删除咪唑洗脱缓冲液。计算蛋白浓度和纯化蛋白质在-80 ° C，直到使用的存储。 </li></ol></li></ol> 2。小鼠免疫及样品采集<ol><li>预温西格玛辅助系统（SAS）40-45 ° C，符合制造商的说明。加入1每ml小瓶PBS调匀。 </li><li>根据表2中的协议，准备蛋白佐剂乳化。移液器计算为1.5毫升管rRBD蛋白质。加入同等体积的SAS辅助大力为2-3分钟，以形成乳化液管和涡。 提示：在表2中列出的卷是一个鼠标。调整卷，根据实际使用的鼠标号码。对于每个组，每一种疫苗所需的蛋白质和辅助混合在一起。疫苗接种，以确保准确性，始终准备一个额外的样品。 <table border="1"><tbody><tr><td> 集团 </td><td> 第1次疫苗 （200μL/鼠标） </td><td> 第2 次疫苗 （200μL/鼠标） </td><td> 第3 次疫苗 （200μL/鼠标） </td></tr><tr><td> rRBD蛋白</td><td> 20μg蛋白在PBS （100μL）+ 100μL的SAS </td><td> 10μg蛋白在PBS（100μL）+ 100μL的SAS </td><td> 10μg蛋白在PBS（100μL）+ 100μL的SAS </td></tr><tr><td> PBS对照</td><td> 100μL的PBS + 100μL的SAS </td><td> 100μL的PBS + 100μL的SAS </td><td> 100μL的PBS + 100μL的SAS </td></tr></tbody></table> 表2。小鼠免疫协议</li><li>皮下注射免疫总理的雌性BALB / c小鼠（4-6周龄，5只/组），并加强与rRBD和SAS两次如表2所示。 PBS作为对照组。皮下注射的网站通常选择的肩胛骨之间的皮肤松弛。此外，腹部腹面是常用的，因为它更容易注入，并可能会观察到从注射部位的任何泄漏。当使用重复剂量的疫苗，它是很容易来选择不同的注射部位，防止潜在的局部皮肤反应。 </li><li>流血通过眼窝每个接种疫苗的免疫接种后10天前与麻醉剂，并在56 ° C下30分钟热血清灭活补体小鼠。商店小鼠血清于-20 ° C，直到使用。 </li></ol> 3。中和检测，​​使用Pseudovirus中和试验<ol><li>准备非典pseudovirus <ol><li> 293T细胞分裂100毫米的组织培养培养皿（2 × 10 6细胞/皿）转染前16小时，和成长细胞如上。 </li><li>准备转染试剂，如表1所示。联合转染质粒编码SARS冠状病毒S蛋白和环境保护缺陷，质粒编码荧光素酶表达的HIV - 1的基因组（pNL4 3.luc.RE）使用磷酸钙转染试剂。 </li><li>更换新鲜DMEM培养液10毫升含10％胎牛血清和1％的P / S 8-10小时后转染介质。收集上清含有非典pseudovirus 72小时后转。 </li><li>通过0.45μm滤膜过滤pseudovirus。分装和储存在-80 ° C，直到使用。 </li></ol></li><li>非典pseudovirus中和试验<ol><li>分割293T细胞表达在SARS病毒受体ACE2的（ACE2/293T）10 4 cells/100μL/孔，96孔组织培养板16小时前感染。 </li><li>稀释2倍SARS pseudovirus ACE2/293T细胞检测病毒滴度。 </li><li>串行稀释的小鼠血清中的96孔组织培养板，加等体积滴定非典pseudovirus。在37 ° C，1小时Preinc​​ubate板</li><li>孵育后，加入100μL血清pseudovirus混合ACE2/293T细胞，并继续增长，细胞在37 ° 5％的CO 2的彗星。加入新鲜的DMEM培养24 h后。 </li><li>彻底清除板从培养上清感染后72 h。添加1X荧光素酶细胞培养裂解试剂（60μL/孔），并不断摇动板在室温下1-2 h促进细胞裂解。 </li><li>转移细胞裂解液（50μL/孔）进入光度计板（Microfluor 96孔板）。加入荧光素酶检测系统中的荧光素酶底物（50微升/孔），超384光度计检测相对荧光素酶的活性。 </li><li>非典pseudovirus中和效价和目前计算为50％的中和抗体滴度（NT 50）6 。 </li></ol></li></ol>

<ol>
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细胞密度是一个重要因素，影响磷酸钙为基础的转的疗效。根据我们的经验，低于70％的细胞汇合带来最好的结果。因此，为了提高转染效率，建议保持在约50-70％汇合，细胞密度。磷酸钙转染法一般被认为与其他转染方法，如脂质转染16相比，效率较低。然而，在这个协议中，我们使用一个修改过的磷酸钙转染法，即常数和慢转的解决方案中的一个旋涡混合确保适当的大小和形状形成沉淀，从而提高转染效率。

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4*7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2429</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2*2H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C5080</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12430</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>OPTI-MEM I Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836170001</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Superflow</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>30450</td>
<td>&nbsp;</td>
<tr>
<td>Sigma adjuvant system</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6322</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase assay system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1501</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase cell culture lysis reagent (5X)</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1531</td>
<td>&nbsp;</td>
<tr>
<td>Amicon Ultra - 15</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC901024</td>
<td>&nbsp;</td>
<tr>
<td>Microfluor 96-well plates</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>7905</td>
<td>&nbsp;</td>
<tr>
<td>Ultra 384 luminometer</td>
<td>&nbsp;</td>
<td>Tecan Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection

基于其安全性和诱导抗感染的免疫反应的能力，亚单位疫苗已被用来作为候选人为多种病原体 1-3 。由于哺乳动物细胞系统的能力，从而形成正确折叠和糖基化蛋白质翻译后修饰，在哺乳动物细胞中表达的重组蛋白都表现出了极大的潜力，以保持较高的抗原性和免疫原性4-6。 自2004年以来，虽然没有发现新病例的SARS疫情的是一个恒定的威胁，因此，对SARS冠状病毒疫苗的发展是一个审慎的预防的步骤，并应进行。 RBD的SARS - CoV的S蛋白在受体结合，诱导特异性中和抗体对病毒感染7-9扮演重要的角色。因此，在这个协议中，我们描述发展RBD的亚单位疫苗，对SARS的新方法。简单地说，RBD的重组蛋白（rRBD）在培养的哺乳动物293T细胞培养上清表示，获得正确的构型和高免疫原性6正确折叠的蛋白质。的RBD的重组质粒转染的细胞，然后使用一些修改一个磷酸钙转染法6,10。 11,12与脂质转染法相比，这个修改过的磷酸钙转染法是更便宜，更容易处理，有可能达到疗效高，一旦形成一个合适的大小和形状的转复杂13,14。最后，在协议中引入综合症pseudovirus中和试验，用于检测rRBD蛋白免疫小鼠血清中和活性。此实验是比较安全的，不涉及传染性的SARS冠状病毒，并可以不执行了一个生物安全实验室 15的要求。 这里所描述的协议也可以用来设计和研究I类融合蛋白的重组亚单位疫苗，对其他病毒，例如，艾滋病毒，呼吸道合胞病毒（RSV），埃博拉病毒，流感病毒，以及尼帕和Handra病毒。此外，产生pseudovirus和随后建立pseudovirus中和试验方法可以适用于所有这些病毒。

Watch this Scientific Journal Video about Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection at JoVE.com

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

这一协议描述为抗击非典研究重组受体结合域（RBD）为基础的亚单位疫苗的一般程序。它包括RBD的蛋白在293T细胞中，免疫与RBD的小鼠，检测小鼠血清中和使用建立非典pseudovirus中和试验活动的转染和表达方法。

协议RBD - SARS疫苗的重组蛋白的制备，动物疫苗接种和中检测

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

小印度蒙古狼的异苯抗酸模拟分析(赫皮斯特奥罗普塔图斯)塞拉,用作口服狂犬病疫苗接种生物标记

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

使用基于HiBiT的生物报告器检测SARS-CoV-2受体结合域抗体

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

使用报告基因表达重组SARS-CoV-2对K18 hACE2转基因小鼠的病毒感染进行实时成像和定量

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...