A novel method for synthetic vaccine construction based on protein assembly

Liu, Zhida; Zhou, Hang; Wang, Wenjun; Tan, Wenjie; Fu, Yang-Xin; Zhu, Mingzhao

Assembly of synthetic DEC205-targeted vaccine using optimized SpyTag/SpyCatcher system. A full vaccine usually consists of at least an immunoregulatory functional unit in addition to the antigen. The modified single-chain antibody aDEC205-SpyTag was used as a functional unit in the present study. The OVA 8 -TBEV ED3 DNA sequence, which encodes model antigens including both a CD8 Tcell epitope (ovalbumin 257-264 ) and a B-cell epitope (TBEV ED3), was genetically fused to the C-terminus of SpyCatcherDN (Sc-OVA 8 -ED3). Conjugation of aDEC205-SpyTag and Sc-OVA 8 -ED3 would result in a fully functional vaccine ( Figure 2a ). aDEC205-SpyTag was expressed in FreeStyle TM 293-F cells and purified by Protein A chromatography ( Figure 2b , Lane 1). The Sc-OVA 8 -ED3 fusion protein was expressed in E. coli and purified by Ni-NTA chromatography ( Figure 2b , Lane 2). The covalent binding reaction was tested under different conditions and with different molar ratios. A 151.5 molar ratio of aDEC205-SpyTag:Sc-OVA 8 -ED3 at 4uC for 2 h was found to give rise to an optimal binding efficiency for the proteins. SDS-PAGE analysis showed that more than 90% of the input aDEC205-SpyTag was conjugated ( Figure 2b , Lane 3). The synthetic DEC205-targeted vaccine (aDEC205-Sc-OVA 8 -ED3) was then purified by Protein A chromatography, with a purity above 90% (Figure 2b , Lane 4).