gM囊泡的表征鉴定及成分分析

Characterization and component analysis of gM vesicles

摘要:

马疱疹病毒-1型(equine herpesvirus-1, EHV-1) gM基因的羧基端可诱导哺乳动物细胞产生能够负载或展示靶蛋白质的囊泡(gM囊泡)，有望应用于药物靶向递送、基因治疗及囊膜化病毒样颗粒疫苗研发等领域。为了研究该囊泡的形成机制、结构构成及生物学功能，本研究使用免疫亲和技术纯化gM囊泡，通过透射电子显微镜(transmission electron microscope, TEM)、纳米颗粒跟踪分析技术(nanoparticle tracking analysis, NTA)对纯化的gM囊泡的形态、数量及Zeta电位进行分析；同时，利用蛋白质质谱技术(liquid chromatography-tandem mass spectrometry, LC-MS/MS)鉴定gM囊泡的结构蛋白，并对蛋白质鉴定结果进行基因本体(gene ontology, GO)功能注释和KEGG (Kyoto encyclopedia of genes and genomes)代谢通路分析。结果显示，gM囊泡直径分布在100?300 nm之间，其平均直径约(151.3±5.7) nm；囊泡Zeta电位为?32.579 mV；纯化囊泡总量可达3.85×10⁵ particles/mL细胞；利用蛋白质质谱技术共鉴定出416种蛋白质，去除无注释的蛋白后保留396种蛋白质用于后续分析；通过GO及KEGG分析，gM囊泡共参与了4条与囊泡形成及运输过程相关的代谢通路，分别为囊泡介导的运输、内吞作用、内质网与高尔基体间的运输和内质网中的蛋白质加工。本研究通过蛋白质谱鉴定分析从蛋白质水平上揭示了gM囊泡的形成机制，为gM囊泡在工程化改造、胞内调控机制、药物递送靶向治疗等领域的研究及应用奠定基础。

Abstract:

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.