A novel method for synthetic vaccine construction based on protein assembly

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

Intracellular cytokine staining assay. Naïve WT C57BL/6 mice were subcutaneously immunized twice with 30 pmol of aDEC205-Sc-OVA 8 -ED3 or Sc-OVA 8 -ED3, along with 30 mg CpG1826 (Invitrogen Life Technologies, Beijing, China) and 30 mg Poly I:C (InvivoGen, San Diego, CA, USA) as an adjuvant, at a 7-day interval. Five days after the secondary vaccination, the spleens of the immunized mice were harvested and processed into single-cell suspensions. Splenocytes (1 3 10 6 cells/well in triplicate) were restimulated in U-bottom 96-well plates with 5 mg/ml OT1 peptide (SIINFEKL) (ChinaPeptides, Suzhou, China) for 6 h in the presence of brefeldin A (5 mg/ml) at 37uC with 5% CO 2 . After restimulation, the cells were first surface stained with APC-conjugated anti-mouse CD8a antibody (53-6.7) before fixation/ permeabilization and intracellular staining for IFNc (XMG1.2). All of the reagents and antibodies were purchased from eBioscience, and the manufacturer's protocol was followed for the surface and intracellular staining.