Fontana Diego, Etcheverrigaray Marina, Kratje Ricardo, Prieto Claudio
Cell Culture Laboratory, Biochemistry and Biological Sciences School, Universidad Nacional del Litoral, Bv. Pellegrini 2750, Santa Fe, 3000, Argentina.
Biotechnological Development Laboratory, Biochemistry and Biological Sciences School, Universidad Nacional del Litoral, Santa Fe, Argentina.
Methods Mol Biol. 2016;1403:155-66. doi: 10.1007/978-1-4939-3387-7_7.
Rabies is a viral infection of the central nervous system for which vaccination is the only treatment possible. Besides preexposure, vaccination is highly recommended for people living in endemic areas, veterinarians, and laboratory workers. Our group has developed rabies virus-like particles (RV-VLPs) with immunogenic features expressed in mammalian cells for vaccine applications. In this chapter the methods to obtain and characterize a stable HEK293 cell line expressing RV-VLPs are detailed. Further, analytical ultracentrifugation steps to purify the obtained VLPs are developed, as well as western blot, dynamic light scattering, and immunogold electron microscopy to analyze the size, distribution, shape, and antigenic conformation of the purified particles. Finally, immunization protocols are described to study the immunogenicity of RV-VLPs.
狂犬病是一种中枢神经系统的病毒感染，对此接种疫苗是唯一可行的治疗方法。除暴露前预防外，强烈建议生活在狂犬病流行地区的人群、兽医和实验室工作人员接种疫苗。我们的团队已开发出具有在哺乳动物细胞中表达的免疫原性特征的狂犬病病毒样颗粒（RV-VLPs）用于疫苗应用。在本章中，详细介绍了获得和鉴定表达RV-VLPs的稳定HEK293细胞系的方法。此外，还开发了用于纯化所得VLPs的分析超速离心步骤，以及用于分析纯化颗粒的大小、分布、形状和抗原构象的蛋白质印迹法、动态光散射法和免疫金电子显微镜法。最后，描述了免疫方案以研究RV-VLPs的免疫原性。
