Preparation of PrP-specific Polyclonal Antibody via Immunization of PRNP-knockout Mice with Recombinant Human PrP Protein

Objective The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrPSc in the brain tissues of suspected cases.Thus,developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.Methods We prepared a PrP-specific polyclonal antibody(pAb P54)in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23–231.Thereafter,we verified that pAb in Western blot,immunohistochemistry(IHC),and immunofluorescent(IFA)assays.Results Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein,normal brain PrPC from healthy rodents and humans,and pathological PrPSc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases.The electrophoretic patterns of brain PrPC and PrPSc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies.Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54.IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139 A-infected mice and 263 K-infected hamsters.IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.Conclusion The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and,thus,may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Global Profiles of Acetylated Proteins in Brains of Scrapie Agents 139A- and ME7-Infected Mice Collected at Mid-Early, Mid-Late, and Terminal Stages

Objective To describe the global profiles of acetylated proteins in the brains of scrapie agents 139Aand ME7-infected mice collected at mid-early,mid-late,and terminal stages.Methods The acetylated proteins from the cortex regions of scrapie agent(139A-and ME7)-infected mice collected at mid-early(80 days postinfection,dpi),mid-late(120 dpi),and terminal(180 dpi) stages were extracted,and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry.The proteins in the infected mice showing 1.5-fold higher or lower levels than that of agematched normal controls were considered as differentially expressed acetylated peptides(DEAPs).Results A total of 118,42,and 51 DEAPs were found in the brains of 139A-80,139A-120,and 139A-180dpi mice,respectively.Meanwhile,390,227,and 75 DEAPs were detected in the brains of ME7-80,ME7-120,and ME7-180 dpi mice,respectively.The overwhelming majority of DEAPs in the mid-early stage were down-regulated,and more portions of DEAPs in the mid-late and late stages were up-regulated.Approximately 22.1%(328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated.Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39(80dpi),13(120 dpi),and 10(180 dpi) significantly changed pathways in 139A-infected mice.Meanwhile,55,25,and 18 significantly changed pathways were observed in the 80,120,and 180 dpi samples of139A-and ME7-infected mice(P < 0.05),respectively.Six pathways were commonly involved in all tested samples.Moreover,many steps in the citrate cycle(tricarboxylic acid cycle) were affected,represented by down-regulated acetylation for relevant enzymes in the mid-early stage and upregulated acetylation in the mid-late and late stages.Conclusion Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection,showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.

Analysis of the Electrophoretic Profiles of Prion Protein in Carcinous and Pericarcinous Lysates of Six Different Types of Cancers

Objective To find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric,colon,liver,lung,thyroid,and laryngeal cancers.Methods Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot were used to test the amounts and electrophoretic patterns of total Pr P and the tolerance of PK(protease K)digestion among six various cancer tissue types.Results A mass of Pr P signals with a large molecular weight were identified in the homogenates of peripheral tissues.The amounts and electrophoretic patterns of total Pr P did not differ significantly between carcinous and pericarcinous tissues.Pr Ps in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types.Conclusions The study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar.Unlike Pr P-specific immunohistochemical assay,evaluation of Pr P electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.