Nanoparticle orientationally displayed antigen epitopes improve neutralizing antibody level in a model of porcine circovirus type 2

Ding, Peiyang; Zhang, Teng; Li, Yafei; Teng, Man; Sun, Yaning; Liu, Xiao; Chai, Shujun; Zhou, Enmin; Jin, Qianyue; Zhang, Gaiping

The Cap protein was expressed in E. coli BL21 (DE3) and purified by affinity chromatography on a Ni 2 + -NTA column. SDS-PAGE and western blotting results showed that the Cap protein was successfully expressed and purified with an apparent molecular mass of approximately 26 kDa (Figure 2A) . The Cap protein reacted with anti-PCV2 Figure 2B ). The mean diameter of AuNPs was 23.2 nm (~23 nm) ( Figure 2C and E). After conjugation of Cap protein, the mean diameter increased to 42.2 nm (~42 nm) ( Figure 2D and E), the absorbance wavelength shifted to 529 nm, and the zeta potential had a positive shift from -47.9 mV to -34.7 mV ( Figure 2F ), indicating stable conjugation between Cap protein and AuNPs. Cap-AuNPs aggregated when they were lyophilized and resuspended, with particle size increasing to 182 nm and zeta potential increasing to -20.8 mV ( Figure 2E and F). The FTIR spectrum ( Figure 2G ) showed a weak peak near 2,550 cm -1 region, which virtually confirmed the presence of thiol (S-H) molecules in the sole cysteine molecule of Cap protein; however, the peak disappeared when AuNPs were coated with Cap protein ( Figure 2G ) indicating that S-H was changed to a new bond. This coincides with that reported by Aryal et al, who discovered the absence of S-H representative peak using FTIR spectrometer and the constitution of new Au-S bond when conjugating cysteine with AuNPs. The thiol molecules of cysteine could directly bind to AuNPs as the covalent Au-S bond is strongly favored. In this study, AuNPs were exploited as the support structure and conjugated with Cap through the Au-S bond. 35 9F4 and 6A5 are mAbs with neutralizing ability that specifically recognize neutralizing epitopes, which are located on the outer surface of the Cap protein. However, a His tag located at the N-terminus of Cap protein replaced the nuclear localization sequence of Cap protein, which is located on the inner face of Cap protein. Furthermore, 8A10 can recognize the decoy epitope Cap (169-180), which was buried when forming VLPs. ELISA results showed that Cap-AuNPs were more easily recognized by 9F4 and 6A5 than Cap; on the contrary, Cap was more easily recognized by anti-His-tag mAb and 8A10 than Cap-AuNPs (Figure 3) . These results indicate that the epitopes on the inner face of Cap protein were hidden, but the neutralizing epitopes located on the outer surface were exposed when Cap protein was conjugated to AuNPs via the Au-S bond.