Basic Research Of The Vaccine Of PRNP Vectors With Ubiquitin Or The Lysosome-targeting Signal

Objective: To evaluate PrP expression characteristic and the basic data of PRNP nucleic acid vaccine with ubiquitin or the lysosome-targeting signal.Method: The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector, that is, pcDNA3.1-UbPrP and pcDNA3.1-PrPLⅡwere constructed. The expression characteristics of PrP with two signals were evaluated by western blot and the localization was observed by indirect immune fluorescence. The first four group mice were immuned by DNA vaccine. Each group was made of four mice, and each mouse received three DNA immunise at 14-day intervals. The other four group mice were immuned by DNA vaccine and boosted by recombinant prion protein. The immune responses of different groups were analyzed in 2 weeks after the last immunization. Specificity and sensitivity of antibodies were detected by both ELISA and Western blotting. Lymphocyte were collected and stimulated with recombinant human PrP protein from different groups, IFN-r was detected by ELISPOT.Result: The protein expressed by pcDNA3.1-UbPrP and pcDNA3.1-PrPLⅡwith ubiquitin and lysosome-targeting signal can be recognized by prion-specific antibody. The protein has three glycosylation molecules form as native PrP. PrP with ubiquitin was degraded gradually with time extension, whereas quantity of PrP with lysosome signal reduced in 48h after transfection. The protein with two location signals can direct fusion prion proteins into cytoplasm. The ELISPOT result show, there are 56,50 SFCs/106/PBMC in two mice of the UbPrP group respectively, 215,50SFCs/106/PBMCs in two mice of the UbPrP+rPrP group respectively, 113.5 SFCs/106/PBMCs in one mouse of the PrP+rPrP group. Lymphocyte from the other mice do not secreated PrP-specific IFN-γ. The ELISA results found that the titer of serum from mice immunized with a plasmid encoding both ubiquitin-tagged and lysosome-targeting signal PrP were above 1:400. But P/N levels of the serum titers from mice immunized with pcDNA3.1-PrP or pcDNA3.1 were below 2.1 The titer of serum from the protein boost group were much higher than the DNA immune group. The titers were above 1:51200 in the rPrP group, and the titers of mice with plasmid Primary Immunizied and protein boosted were above 1:102400. Antibodies induced after immunized with pcDNA3.1-UbPrP, pcDNA3.1-PrPLⅡ, pcDNA3.1-UbPrP+rPrP, pcDNA3.1-PrP+rPrP, pcDNA3.1-PrPLⅡ+rPrP could recognize the recombinant PrP proteinby Western bloting. The pcDNA3.1-UbPrP can breaked immune tolerance and enhanced both humoral and cellular responses against human prion protein.Conclusion: The PRNP vaccine with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. The vaccine with ubiquitin can break immune tolerance against the prion protein in wild-type mice, resulting in the induction of PrP-specific antibody and T-cell responses.