具核梭杆菌外膜囊泡蛋白组分和抗原性分析及对宿主细胞毒性研究

Protein composition and antigenicity analysis of outer membrane vesicles of Fusobacterium nucleatum and study on host cytotoxicity

目的 研究具核梭杆菌(Fusobacterium nucleatum,Fn)外膜囊泡(Outer membrane vesicle,OMV)蛋白质组学特征及其对宿主细胞的毒性作用。方法 使用梯度离心法从Fn的培养上清液中分离纯化出OMV (Fn-OMV),通过中文名称(Nano-LC/MS/MS)分析鉴定Fn-OMV携带的蛋白质;利用GO数据库进行分类,并通过KEGG注释通路进行分类和分析。通过流式细胞术检测Fn-OMV处理人白血病T细胞Jurkat细胞和小鼠巨噬细胞RAW264.7后细胞凋亡的情况,同时使用Fn活菌作为对照;通过细胞增殖毒性实验(CCK8)探究Fn-OMV对人结肠癌RKO细胞增殖的影响。结果 通过革兰染色观察确定Fn形态,粒径分析所提取的Fn-OMV平均大小为126.5 nm,质量良好。Nano-LC/MS/MS结果表明Fn-OMV共携带509个蛋白,占Fn全菌蛋白的25%,其中主要的细菌外膜蛋白有10个,均具有良好的抗原性和多个抗原决定簇。通过GO分类发现Fn-OMV携带大量分子功能相关蛋白,经KEGG注释通路分析包含蛋白数目最多的前10个通路为:抗生素合成,碳代谢,核糖体,氨基酸合成,嘌呤代谢,氨酰tRNA合成,丁酸盐代谢,糖酵解/糖异生,丙酮酸代谢,氨基酸和核苷酸糖代谢。流式细胞术结果表明Fn活菌可促进Jurkat细胞和RAW264.7细胞凋亡,但Fn-OMV对二者凋亡无显著作用。Fn-OMV对RKO细胞的增殖无显著作用。结论 Fn-OMV携带的细菌外膜蛋白有10个,均具有良好的抗原性和多个抗原决定簇,与Fn相比,其对细胞凋亡和增殖无显著作用。因此,本研究对基于OMV的Fn疫苗设计提供了基础信息,具有潜在的应用价值。

Objective To study the proteomic characteristics of Fusobacterium nucleatum(Fn) outer membrane vesicle(OMV) and to investigate its role in host cells.Methods OMV were isolated and purified from the culture supernatant of Fn by gradient centrifugation.Proteins carried by Fn-OMV were identified by Nano-LC/MS/MS analysis.The GO database was used for classification,and KEGG annotated pathways were classified and analyzed.The apoptosis effect of Fn-OMV on Jurkat and RAW264.7 cells was examined by flow cytometry analysis.Live Fn was used as control.RKO cells proliferation was determined by CCK8 assay after been treated with Fn-OMV.Results The morphology of Fn was confirmed by Gram staining.the mean particle size of Fn-OMV was 126.5 nm,and it exhibited good quality.Nano-LC/MS/MS analysis identified Fn-OMV carrying 509 proteins,accounting for 25% of the total bacterial protein of Fn,among which there are 10 main bacterial outer membrane proteins,and the outer membrane proteins carried have good antigenicity and many antigenic determinants.Using GO classification,it was found that Fn-OMV carries a large number of proteins related to molecular functions.The KEGG annotation pathway analysis showed that the top ten pathways with the largest number of proteins were:antibiotic synthesis;carbon metabolism;ribosome;amino acid synthesis;purine metabolism;aminoacyl tRNA synthesis;Butyrate metabolism;glycolysis/gluconeogenesis;pyruvate metabolism;amino acid and nucleotide sugar metabolism.Flow cytometry showed that Live-Fn could promote the apoptosis of Jurkat cells and RAW264.7 cells,but Fn-OMV had no apoptosis effect on these cells.Besides,Fn-OMV did not effect RKO cells proliferation.Conclusion There are 10 outer membrane proteins of bacteria carried by Fn-OMV,all of them have good antigenicity and multiple antigenic determinants.Compared with Live-Fn,Fn-OMV did not effect cells apoptosis and proliferation.Therefore,this study provides basis information for the design of Fn-OMV vaccine which has potential application value.