Optimized exosome isolation protocol for herpes simplex virus type 1 infected cells
10.3760/cma.j.cn112866-20210323-00052
- VernacularTitle:高效分离1型单纯疱疹病毒感染细胞外泌体的研究
- Author:
Li ZHANG
1
;
Jiao WANG
;
Zhan WANG
;
Ying LI
;
Hui WANG
;
Jingdong SONG
;
Hongtu LIU
Author Information
1. 中国疾病预防控制中心病毒病预防控制所，北京 102206
- Keywords:
Herpes simplex virus type 1;
HEp-2 cells;
Exosomes;
Isolation;
Purification
- From:
Chinese Journal of Experimental and Clinical Virology
2021;35(6):619-625
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish and optimize the isolation scheme of exosomes from herpes simplex virus type 1 (HSV-1) infected HEp-2 cells.Methods:The supernatant of HSV-1 infected HEp-2 cells were collected and pretreated with differential centrifugation and filtration, then purified by total exosome isolation (TEI) in combination with size-exclusion chromatography (SEC); and enriched the exosome with ultrafiltration. Western blotting, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were performed to characterize the purified exosomes.Results:The purified exosomes from HEp-2 cells with or without HSV-1 infection showed positive signals of exosome-specific markers (TSG101 and CD9), and the expression of virus protein gB, VP16 and ICP5 were detected in exosomes derived from HSV-1 infected cells. The purified exosomes from HEp-2 cells with or without HSV-1 infection showed a typical round-shaped morphology under TEM, with a mean diameter of 50~150 nm; meanwhile, no typical HSV-1 virus particles were detectable. The peak diameter of purified exosomes was 112.2 nm, furthermore, the peak concentration of exosomes from HEp-2 control cells was 3.4×10 9 particles/ml, while the peak concentration of exosomes from HSV-1 infected cells was 4.1×10 9 particles/ml. Conclusions:We accomplished an efficient and optimized approach for the isolation and purification of exosomes from HSV-1 infected cells.
