[Characterization and component analysis of gM vesicles]

The carboxyl-terminal domain of the equine herpesvirus-1 (EHV-1) gM gene has been shown to induce the production of gM vesicles capable of loading or displaying target proteins in mammol/Lalian cells. These vesicles show promising potential for applications in targeted drug delivery, gene therapy, and the development of enveloped virus-like particle (eVLP) vaccines. However, the biogenesis, structural composition, and biological functions of gM vesicles remain poorly understood. To address this, we employed immol/Lunoaffinity capture to purify gM vesicles and characterized their morphology, concentration, and zeta potential by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). In parallel, the protein composition of gM vesicles was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by functional annotation via GO (gene ontology) and pathway analysis using KEGG (Kyoto encyclopedia of genes and genomes). The results demonstrated that gM vesicles exhibited a diameter range of 100‒300 nm, an average diameter of (151.3±5.7) nm, a zeta potential of ‒32.579 mV, and a yield of approximately 3.85×10⁵ particles/mL. A total of 416 proteins were identified by LC-MS/MS. Following the exclusion of unannotated proteins, 396 proteins were remained for subsequent GO and KEGG analyses. The results revealed that gM vesicles were involved in four metabolic pathways associated with vesicle formation and trafficking: vesicle-mediated transport, endocytosis, endoplasmic reticulum-Golgi transport, and protein processing within the endoplasmic reticulum. This study elucidates the formation mechanism of gM vesicles at the protein level, thereby establishing a foundation for their future applications in various fields, such as modification of gM vesicles, regulation of intracellular vesicle dynamics, drug delivery, and targeted therapy.