| Nanobodies(or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from the heavy chain antibodies of camels and sharks. Their comparatively small size, high stability, poor immunogenicity, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and diagnostic purposes. Here we created the immune Camelus Bactrianus VHH yeast two-hybrid(Y2H) library. Using as an example the porcine circovirus type 2(PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. prokaryotic expression of these VHHs and subsequent functional verification by ELISA revealed that several of these VHHs had better bioactivity prior to the positive antiserum in commercial diagnostic kit.Based on our previous study, we selected a VHH with the highest bioactivity for further post modification. First, the homodimer of the selected VHH was generated by fusion the two copies of the VHH with a flexible linker. Second, a translation enhanced element(TEE) was introduced by fusion with the N terminus of the VHH homodimer to increase protein production. Third, the VHH homodimer was further biotinylated by fusion the VHH homodimer with avidin-tag at C terminus and expression in recombinant E.coli for protein bioinylation. The result indicated that the bioactivity of VHH was expectedly improved by dimerization and biotinylation.Biotin-avidin has been a signaling cascade system extensively used in recent years. And the antigen-antibody affinity was greatly developed by generating homodimer of VHH. Furthermore, the introduction of TEE could improve the production of exogenous protein in E.coli. Therefore, this study suggested that the modification of biotinylated bivalent VHH is a powerful strategy to improve the bioactivity of VHH against PCV2 Cap protein. |
