| Objective:To break immune tolerance against the prion protein, we used PRNP DNA vaccines including with ubiquitin, lysosome and endoplasmic reticulum-targeting signal to immunize the wide-type mice, expecting to get better humoral and cellular responses, so that we can get some research data on prion diseases.Methods:(1) The protein expression of PrP DNA vaccines after transient transfection into HeLa cells was identified by PrP specific Western Blot. (2) The inoculation strategy is intramuscular injection of DNA vaccine combined with enhancing by hypodermic injection of recombinant protein.42 mice was set into 6 groups randomly(7mice/group) including pcDNA3.1 as the control, pcDNA3.1 with protein enhancing, pcDNA3.1-PrP, pcDNA3.1-UbPrP, pcDNA3.1-PrPLII and pcDNA3.1-PrP-ER enhanced by protein. The DNA vaccine plasmids were diluted into 1μg/μl, and then injected 50μl into the gastrocnemius of mice each side every time, namely, 100μg every time a mouse. After three DNA immunizations, the belly hypodermic injection of recombinant full-length PrP (PrP23-231) was operated twice to boost the immunization at interval of two weeks. And two weeks after the last immunization, the serum and spleen cells of the mice were collected, and then serum IgG and spleen cells were detected by ELISA and ELISPOT assay respectively.Results. (1) After 36h and 48h of DNA vaccines transfection, PRNP DNA vaccines could express PrP protein excepting pcDNA3.1. But the quantity and the characteristic of the protein's expression were not similar each other. DNA vaccine of pcDNA3.1-PrP expressed much more protein than any other groups within 48h transfection. Furthermore, the protein expression after 48h transfection was more than that of after 36h. The prion proteins expressed by pcDNA3.1-UbPrP were mainly composed with disglycosylation, and with the time extension to 48h, the protein of disglycosylation degradated more quickly. Monoglycosylaton PrP protein was encoded by pcDNA3.1-PrPLII vector primarily. For pcDNA3.1-PrPER vaccine, the protein of disglycosylation is little, while the nonglycosylated protein is increased significantly. (2) The detection of the serum IgG antibodies. The DNA vaccine of pcDNA3.1-PrP, pcDNA3.1-UbPrP, pcDNA3.1-PrPLII, pcDNA3.1-PrPER could induce PrP-specific antibodies and the titers were up to 6400,6400,6400 and 3200 accordingly. And pcDNA3.1 enhanced with recombinant PrP can also induced PrP-specific antibodies at the title of 200. Moreover, all antiserum from various PRNP vaccines could identify the recombinent PrP protein expressed in E. coli specifically. (3) Detection of cellular responses by ELISPOT assay. Splenocytes of mice inoculating with various PRNP vaccines were stimulated to observe the celluar immunity effect by PrP23-90, PrP91-231 and PrP23-231 protein. The results showed that SFCs from mice inoculating with pcDNA3.1-PrP-ER, pcDNA3.1-PrP and pcDNA3.1-UbPrP groups stimulated by PrP23-90 were 720/106 splenocytes,538/106 splenocytes and 482/106 splenocytes. 483/106 splenocytes,274/106 splenocytes and 261/106 splenocytes of mice inoculating with pcDNA3.1-PrPER, pcDNA3.1-PrPLII and pcDNA3.1-UbPrP groups were induced respectively by PrP23-231 protein stimulation. While the SFCs stimulated by PrP91-231 was less than 50 among every 106 splenocytes from all groups. The results suggest us that the epitope of PrP T cells response was located at the N-terminal 23-90aa of Prion protein.Conclusion:The PRNP vaccines including ubiquitin, lysosome and endoplasmic reticulum-targeting signals can break the host tolerance to Prion protein in wild-type mice, and induced better humoral and cellular responses. The PRNP DNA vaccines immunization followed by recombinant protein boosting can improve the level of the immune response to Prion protein at a certain extent.Figure [10] table [10] reference [69]... |
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