کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
4752201 | 1361276 | 2017 | 8 صفحه PDF | دانلود رایگان |
- A preparation process for a novel EV71 vaccine candidate was developed.
- The yield for the fused antigen is about 60Â mg/L culture.
- Fused antigen self-assembled as nanoparticles and elicit a high immune response.
An efficient preparation process for a novel Enterovirus 71 (EV71) vaccine was developed in this paper, which is a fused antigen by connecting the truncated capsid proteins of VP1, VP2 and VP3 into one molecule through flexible peptide linkers and expressed in E. coli as inclusion bodies. The fused protein was purified at denatured state through two-step ion exchange chromatography, with final purity above 95%, host cell proteins below 0.003% and residual DNA less than 50Â ng/mL. During the following refolding and assembling process through dilution, the fused antigen precipitated completely, while the precipitation was efficiently inhibited with 2Â M urea or 0.5Â M arginine as an additive. Size exclusion chromatography analysis indicated the protein formed soluble aggregates with linear or rod-like appearance in transmission electron microscopy. These aggregates transformed into pentamers with a size of 15Â nm at pH 8.0 after the additive removing. Moreover, most of the pentamers assembled as sphere-like particulates about 25-40Â nm after being induced by calcium chloride. High antigen-specific IgG titer was elicited by immunization with the nanoparticles in mouse model. Splenocytes proliferative responses and cytokines analysis indicated this particulate antigen could induce humoral and cellular immune responses. These results lay foundations for developing the fused antigen as an alternative vaccine against hand-foot-and-mouth disease (HFMD) and for the large-scale production for E.coli-based vaccines.
Journal: Biochemical Engineering Journal - Volume 117, Part A, 15 January 2017, Pages 139-146