Name: a "plug-and-display" nanoparticle vaccine platform based on outer membrane vesicles displaying sars-cov-2 receptor-binding domain
Uploaded: 2022-07-25T14:30:00
Description: Watch this Scientific Journal Video about A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain at JoVE.com

这项工作得到了重庆市自然科学基金重点项目（No.cstc2020jcyj-zdxmX0027）和中国国家自然科学基金项目（第31670936号，82041045）。

1. 质粒构建
<ol><li>将编码 SpyCatcher 序列（补充文件1）的DNA插入BamH I和Sal I位点之间的氨苄青霉素耐药pThioHisA-ClyA质粒（参见 材料表），以构建质粒pThioHisA ClyA-SC之前发表的报告20。</li>
	<li>将合成的SpyTag-RBD-Histag融合基因（补充文件1）连接成BamH I和EcoR I位点之间的pcDNA3.1质粒（参见 材料表），以构建质粒pcDNA3....

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+vesicle+vaccines.">Outer membrane vesicle vaccines.</a> Seminars in Immunology. 50, 101433 (2020).</li><li>Toyofuku, M., Nomura, N., Eberl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+and+origins+of+bacterial+membrane+vesicles.">Types and origins of bacterial membrane vesicles.</a> Nature Reviews Microbiology. 17 (1), 13-24 (2019).</li><li>Sartorio, M. G., Pardue, E. J., Feldman, M. F., Haurat, M. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+antibody+responses+to+SARS-CoV-2+in+convalescent+individuals.">Convergent antibody responses to SARS-CoV-2 in convalescent individuals.</a> Nature. 584 (7821), 437-442 (2020).</li><li>Wang, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2:+Structure,+Biology,+and+Structure-Based+Therapeutics+Development.">SARS-CoV-2: Structure, Biology, and Structure-Based Therapeutics Development.</a> Frontiers in Cellular and Infection Microbiology. 10, 587269 (2020).</li><li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunogenicity+of+a+recombinant+tandem-repeat+dimeric+RBD-based+protein+subunit+vaccine+(ZF2001)+against+COVID-19+in+adults:+Two+randomised,+double-blind,+placebo-controlled,+phase+1+and+2+trials.">Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: Two randomised, double-blind, placebo-controlled, phase 1 and 2 trials.</a> The Lancet Infectious Disease. 21 (8), 1107-1119 (2021).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+vaccine-elicited+antibodies+to+SARS-CoV-2+and+circulating+variants.">mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.</a> Nature. 592 (7855), 616-622 (2021).</li><li>Amanat, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+mRNA+vaccination+induces+functionally+diverse+antibodies+to+NTD,+RBD,+and+S2.">SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2.</a> Cell. 184 (15), 3936-3948 (2021).</li><li>Tan, H. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+prime-boost+protein+subunit+vaccine+strategies+against+SARS-CoV-2+in+mice+and+macaques.">Immunogenicity of prime-boost protein subunit vaccine strategies against SARS-CoV-2 in mice and macaques.</a> Nature Communication. 12 (1), 1403 (2021).</li><li>Thapa, H. B., Mueller, A. 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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibody+vaccine+for+pandemic+and+pre-emergent+coronaviruses.">Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.</a> Nature. 594, 553-559 (2021).</li><li>van Saparoea, H. B. V., Houben, D., Kuijl, C., Luirink, J., Jong, W. S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+protein+ligation+systems+to+expand+the+functionality+of+semi-synthetic+outer+membrane+vesicle+nanoparticles.">Combining protein ligation systems to expand the functionality of semi-synthetic outer membrane vesicle nanoparticles.</a> Frontiers in Microbiology. 11, 890 (2020).</li><li>Needham, B. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncating+the+structure+of+lipopolysaccharide+in+Escherichia+coli+can+effectively+improve+poly-3-hydroxybutyrate+production.">Truncating the structure of lipopolysaccharide in Escherichia coli can effectively improve poly-3-hydroxybutyrate production.</a> ACS Synthetic Biology. 9 (5), 1201-1215 (2020).</li><li>Liu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+vesicles+from+flagellin-deficient+Salmonella+enterica+serovar+Typhimurium+induce+cross-reactive+immunity+and+provide+cross-protection+against+heterologous+Salmonella+challenge.">Outer membrane vesicles from flagellin-deficient Salmonella enterica serovar Typhimurium induce cross-reactive immunity and provide cross-protection against heterologous Salmonella challenge.</a> Scientific Reports. 6, 34776 (2016).</li><li>Balhuizen, M. 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为了创建一个“即插即显示”的纳米颗粒疫苗平台，SC融合的ClyA在BL21（DE3）菌株中表达，BL21（DE3）菌株因其在蛋白质表达方面的优势而成为重组蛋白生产中使用最广泛的模型之一24，因此在细菌增殖过程中OMV表面会有足够的SC片段显示。同时，制备了ST融合的靶抗原，用于抗原与OMV之间的化学偶联。该实验方案的优势和未来应用前景主要体现在三个方面。首先，实现不同抗原和?...

作为潜在的疫苗平台，外膜囊泡（OMV）近年来越来越受到关注1，2。OMV主要由革兰氏阴性菌3自然分泌，是由脂质双层组成的球形纳米级颗粒，通常大小为20-300纳米4。OMV含有各种亲本细菌成分，包括细菌抗原和病原体相关分子模式（PAMP），可作为固体免疫增强剂5。得益于其独特的成分、天然囊泡结构和出色的基因工程修饰位点，OMV已被开发用于许多生物医学领域，包括细菌疫苗6，佐剂7，癌症免疫治疗药物8，药物递送载体9和抗菌粘合剂10。
自2020年以来在全球蔓延的SARS-CoV-2大流行对全球社会造成了沉重打击。刺突蛋白（S蛋白）中的受体结合域（RBD）可以与人血管紧张素转换酶2（ACE2）结合，然后介导病毒进入细胞11，12，13。因此，RBD似乎是疫苗发现的主要目标14，15，16。然而，单体RBD的免疫原性较差，其小分子量使免疫系统难以识别，因此通常需要佐剂17。
为了提高RBD的免疫原性，构建了显示多价RBD的OMV。使用OMV显示RBD的现有研究通常将RBD与OMV融合以在细菌中表达18。然而，RBD是一种病毒来源的蛋白质，原核表达可能会影响其活性。为了解决这个问题，使用源自 化脓性链球菌的SpyTag（ST）/SpyCatcher（SC）系统在常规缓冲系统中与OMV和RBD形成共价同价肽19。SC结构域通过生物工程大 肠杆菌以溶细胞素A（ClyA）作为融合蛋白表达，ST通过HEK293F细胞表达系统与RBD表达。OMV-SC和RBD-ST混合并孵育过夜。经超速离心或体积排阻色谱（SEC）纯化后，得到OMV-RBD。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ampicillin sodium</td>
			<td>Sangon Biotech</td>
			<td>A610028</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Countstar</td>
			<td>BioTech</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein quantification Kit</td>
			<td>cwbio</td>
			<td>cw0014s</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touching Imaging System</td>
			<td>Bio-rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Danamic Light Scattering</td>
			<td>Malvern</td>
			<td>Zetasizer Nano S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Cavoy</td>
			<td>Power BV</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Buffers H 10X TBST Buffer</td>
			<td>Sangon Biotech</td>
			<td>C520009</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat pAb to mouse IgG1</td>
			<td>Abcam</td>
			<td>ab97240</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed freezing centrifuge</td>
			<td>Bioridge</td>
			<td>H2500R</td>
			<td></td>
		</tr>
		<tr>
			<td>His-Tag mouse mAb</td>
			<td>Cell signaling technology</td>
			<td>2366s</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sangon Biotech</td>
			<td>A600277</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl beta-D-thiogalactopyranoside</td>
			<td>Sangon Biotech</td>
			<td>A600118</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA His-Bind Superflow</td>
			<td>Qiagen</td>
			<td>70691</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powdered milk</td>
			<td>Sangon Biotech</td>
			<td>A600669</td>
			<td></td>
		</tr>
		<tr>
			<td>OPM-293 cell culture medium</td>
			<td>Opm biosciences</td>
			<td>81075-001</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1 RBD-ST plasmid</td>
			<td>Wuhan genecreat biological techenology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>ZSGB-bio</td>
			<td>ZLI-9061</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine Linear</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained protein ladder</td>
			<td>Thermo</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>pThioHisA ClyA-SC plasmid</td>
			<td>Wuhan genecreat biological techenology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Western Blotting Membranes</td>
			<td>Roche</td>
			<td>03010040001</td>
			<td></td>
		</tr>
		<tr>
			<td>Quixstand benchtop systems (100 kD hollow fiber column)</td>
			<td>GE healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE loading buffer (5x)</td>
			<td>Beyotime</td>
			<td>P0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A100241</td>
			<td></td>
		</tr>
		<tr>
			<td>Supersignal west pico PLUS (enhanced chemiluminescence solution)</td>
			<td>Thermo</td>
			<td>34577</td>
			<td></td>
		</tr>
		<tr>
			<td>Suspension instrument</td>
			<td>Life Technology</td>
			<td>Hula Mixer</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Hitachi</td>
			<td>HT7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>XPN-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet spectrophotometer</td>
			<td>Hitachi</td>
			<td>U-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sangon Biotech</td>
			<td>A610961</td>
			<td></td>
		</tr>
	</tbody>
</table>

a "plug-and-display" nanoparticle vaccine platform based on outer membrane vesicles displaying sars-cov-2 receptor-binding domain

从细菌或病毒中获得的仿生纳米颗粒引起了人们对疫苗研究和开发的极大兴趣。外膜囊泡（OMV）在平均生长过程中主要由革兰氏阴性菌分泌，具有纳米级直径和自佐活性，可能是疫苗递送的理想选择。OMV作为蛋白质、核酸和小分子的多方面递送系统发挥作用。为了充分利用OMV的生物学特性，利用生物工程大 肠杆菌衍生的OMV作为载体，以SARS-CoV-2受体结合域（RBD）为抗原，构建“即插即用”疫苗平台。 化脓性链球菌 中的SpyCatcher（SC）和SpyTag（ST）结构域用于偶联OMV和RBD。质粒转染后，溶细胞素A（ClyA）基因与SC基因一起翻译为融合蛋白，在OMV表面留下反应位点。在常规缓冲系统中将RBD-ST混合过夜后，OMV和RBD之间形成了共价结合。因此，实现了多价显示OMV疫苗。通过用多种抗原代替，OMVs疫苗平台可以有效地显示多种异质抗原，从而有可能快速预防传染病流行。该协议描述了构建OMV疫苗平台的精确方法，包括生产，纯化，生物偶联和表征。

该协议的流程图如图 1 所示。该协议可能是利用OMV作为疫苗平台的一般方法;只需要根据抗原的类型选择合适的表达系统。
图2提供了可行的质粒设计方案。SC基因通过柔性接头与ClyA基因连接，而ST通过His标签基因连接到RBD基因的5'末端进行纯化和验证。蛋白质印迹显示，随着OMV-SC的增加，反应逐渐完成（图3A）...

Watch this Scientific Journal Video about A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain at JoVE.com

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

本协议将外膜囊泡的生物工程描述为“即插即用”疫苗平台，包括生产、纯化、生物偶联和表征。

基于外膜囊泡显示SARS-CoV-2受体结合域的“即插即用”纳米颗粒疫苗平台

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

纳米科学在表面显示抗原后能有效增强抗原特异性免疫应答，该疫苗平台的主要优势是可以实现脱落斑块和显示。换句话说，该平台能够显示各种抗原的方法也可以应用于其他生物纳米颗粒，以显示引擎到纳米尺寸的载体。要进行溶细胞素 A 间谍导管转化，首先将 5 微升血浆溶液加入 50 微升 BL 21 感受态菌株中。轻轻吹气，在冰上冷却 30 分钟。将溶液放入42摄氏度的水浴中90秒，然后立即将混合溶液放在冰上3分钟。向细菌悬浮液中加入500微升luria bertani培养基，并在37摄氏度下以每分钟220转的速度混合培养物一小时。接下来，将所有含有氨苄青霉素的转化luria bertani琼脂平板在37摄氏度下培养过夜。对于OMV间谍捕手的生产，首先从平板中分离出单个菌落到20毫升luria bertani培养基中，并在37摄氏度下以每分钟220转的速度培养过夜。将细菌溶液孵育到两升培养基中，并在37摄氏度下以每分钟220转的速度培养5小时，直到达到对数生长阶段。加入异丙基β D-IPTG，使细菌溶液的最终浓度为0.5毫摩尔，然后在25摄氏度下以每分钟220转的速度培养过夜。接下来，执行OMV间谍捕手纯化。从离心融合细菌溶液开始。用0.22微米膜过滤器过滤上清液，然后使用中空纤维柱浓缩。通过0.22微米膜过滤器过滤浓缩物，然后使用超离心保险丝将其在4摄氏度下以150， 000倍G离心融合两小时，并用移液管丢弃上清液。用PBS重新悬浮沉淀，并将其储存在零下80摄氏度。要进行RBD spytag转染，首先选择合适的真核表达系统，并在恢复后在37摄氏度下以每分钟130转的速度培养细胞过夜。将20微升HEK 293F细胞溶液加入自动细胞计数器中。记录细胞数，将浓度调整为1乘以10至每毫升6个细胞。然后在37摄氏度下以每分钟130转的速度培养细胞4小时。通过0.22微米膜过滤器过滤RBD间谍标签质粒，并将300微克质粒加入细胞培养基中，直到最终体积为10毫升。摇晃 10 秒钟。在水浴中将PEI加热至65摄氏度。将0.7毫升PEI与9.3毫升细胞培养基混合，间歇摇动10秒钟。将质粒溶液添加到PEI溶液中。间歇性摇动混合物 10 秒钟，然后在 37 摄氏度下孵育 15 分钟。将混合物加入280毫升细胞培养基中，并在37摄氏度下以每分钟130转的速度培养五天。接下来，执行 RBD 间谍标签纯化。首先在25摄氏度下以6，000倍G离心融合细胞20分钟，然后使用移液管收集上清液。用两毫升镍NTA琼脂糖填充色谱柱，并用PBS洗涤三次。将咪唑加入细胞上清液中，使终浓度为20毫摩尔，并加载细胞上清液。加入三柱体积的含有20毫摩尔咪唑的PBS进行洗涤，并收集洗涤级分。使用含有低至高浓度咪唑的三柱PBS进行梯度洗脱，每种浓度洗脱两次。为了通过BCA方法测定蛋白质浓度，将标准BSA蛋白质溶液从每毫升2稀释至0.0625毫克，将纯化的OMV间谍捕手和RBD间谍标签稀释10倍。然后以50比1的比例混合BCA工作溶液A和B。加入稀释的蛋白质溶液，并与BCA工作溶液混合。在 37 度下孵育 30 分钟。测量每个孔562纳米处的吸光度，并从标准曲线计算蛋白质浓度。要执行OMV间谍捕手和RBD间谍标签的生物偶联，请以40比1的比例混合OMV间谍捕手和RBD间谍标签和PBS。垂直旋转以在 4 摄氏度下以每分钟 15 转的速度将混合物混合过夜。使用类似于OMV间谍捕手纯化的程序，使用PBS溶液对OMV RBD进行纯化。血浆设计方案表明，spycatcher基因通过柔性接头与cly A基因连接，而spytag通过hys标签基因连接到RBD基因的五个素末端进行纯化和验证。超速离心后，几乎所有未反应的RBD间谍标签都保留在上清液中。与第一次相比，第二次超速离心没有提供进一步的显着优势。OMV捕手的z平均水动力直径为133纳米，而OMV-RBD的平均水动力直径为152.6纳米。这些差异可能是因为RBD在密集显示后增加了OMV粒径。TEM的结果与DLS结果获得的粒度分布一致。用于混合混合物的旋转非常重要。在没有旋转的情况下，反应效率可以低得多。

Research

JoVE Journal

Bioengineering

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

科研

生物工程

Stretching Micropatterned Cells on a PDMS Membrane

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

基于纸张的设备进行隔离外囊泡与表征

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

用于生成多细胞三维球体的 CD 实验室平台

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

用于刺激骨质复变和分析骨骼重塑功能结果的实验室A芯片平台

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

在微加工微流体平台中生成简化的三维芯片皮肤模型

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...