The Norovirus (NoV) P particle (PP) is a subviral particle formed by 24 copies of the protruding (P) domain of the capsid protein. Each P domain has three surface loops that can be used for foreign antigen presentation. Hence, PPs have been demonstrated to be an excellent platform for vaccine development against many pathogens. However, current processes for preparing those chimeric PP vaccines vary and would change the original sequence of the PR. A detailed strategy also has not been reported for inserting a foreign antigen into all three loops. In order to develop a novel method for preparing distinct types of PP-based protein vaccines, we created two restriction enzyme sites (Eagl and KpnI) in the P domain by site-directed mutagenesis without changing its original sequence. A synthesized gene with three copies of the Alzheimer's disease (AD) immunogen A beta 1-6 was then incorporated in loop2 of the P domain. Additionally, a synthesized gene with one copy of A beta 1-6 was inserted into each loop of the P domain. Furthermore, two recombinant proteins PP-3 copy-A beta 1-6-loop2 and PP-1 copy-A beta 1-6-loop123 were successfully purified without affecting PP formation. Particle size analysis and TEM observations demonstrated that the two chimeric P particles were still able to form 24-mer nanoparticles. Moreover, the two chimeric PP-based AD vaccines could both efficiently elicit strong immune responses in the mouse model. In conclusion, we have successfully established a novel method for preparing vaccines based on the NoV PP which would not affect PP sequence and function. (C) 2016 Elsevier Inc. All rights reserved.