JoVE Journal > Bioengineering
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Bioengineering

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

Published: July 25, 2022
doi:
10.3791/64213
, , , , , ,
1National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy,Third Military Medical University

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 质粒构建 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。 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….

Representative Results

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）…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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）。

Materials

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

Keywords: Nanoparticle VaccineOuter Membrane VesiclesSARS-CoV-2 Receptor-binding DomainAntigen DisplayCytolysin A SpycatcherOMV Spycatcher ProductionOMV Spycatcher PurificationRBD Spytag TransfectionEukaryotic Expression System
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Feng, R., Li, G., Jing, H., Liu, C., Xue, R., Zou, Q., Li, H. A “Plug-And-Display” Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain. J. Vis. Exp. (185), e64213, doi:10.3791/64213 (2022).
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