Feng Rang, Li Guo-Cheng, Jing Hai-Ming, Liu Chang, Xue Ruo-Yi, Zou Quan-Ming, Li Hai-Bo
National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University.
National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University;
J Vis Exp. 2022 Jul 25(185). doi: 10.3791/64213.
Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a "Plug-and-Display" vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.
从细菌或病毒中获得的仿生纳米颗粒在疫苗研发中引起了广泛关注。外膜囊泡（OMV）主要由革兰氏阴性菌在正常生长过程中分泌，具有纳米级直径和自佐剂活性，可能是疫苗递送的理想选择。OMV已作为蛋白质、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，将生物工程改造的大肠杆菌来源的OMV用作载体，将严重急性呼吸综合征冠状病毒2（SARS-CoV-2）受体结合域（RBD）用作抗原，构建了一个“即插即显”疫苗平台。化脓性链球菌中的SpyCatcher（SC）和SpyTag（ST）结构域用于连接OMV和RBD。质粒转染后，溶细胞素A（ClyA）基因与SC基因作为融合蛋白进行翻译，在OMV表面留下一个反应位点。在常规缓冲系统中将RBD-ST混合过夜后，OMV与RBD之间形成共价结合。因此，获得了一种多价展示的OMV疫苗。通过替换不同的抗原，OMV疫苗平台可以有效地展示多种异源抗原，从而有可能迅速预防传染病流行。本方案描述了构建OMV疫苗平台的精确方法，包括生产、纯化、生物偶联和表征。
