Conformation based reagents for the detection of disease-associated prion protein.

摘要

Prion diseases are a group of fatal, transmissible neurodegenerative diseases found in both animals and humans. Prion diseases are thought to arise when the normal cellular prion protein, PrPC, undergoes a conformational change from a primarily alpha-helical form into a form rich in beta-sheets and resistant to protease, called PrPSc. Animal forms of prion disease include scrapie in sheep, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) in cattle. In humans, the diseases can take a sporadic, genetic or acquired form. One acquired form of human prion disease is variant Creutzfeldt-Jakob disease (vCJD), which emerged in the United Kingdom in 1995, and is thought to be caused by the consumption of BSE contaminated beef. The only definitive diagnostic techniques available at the present time use protease digested brain tissue.;This thesis sought to develop a panel of diagnostic reagents for prion disease that enable the detection of PrPSc, but not PrP C, without protease digestion. We characterized two short peptide sequences, based on the Kringle domains of the serine protease plasminogen, for binding to PrPSc, following reports that plasminogen has PrPSc binding ability. The short peptides bound to all forms of PrPSc, both animal and human, and binding was retained in PrPSc-spiked human plasma. In addition, a panel of PrP binding DNA oligonucleotides, called aptamers, isolated through the SELEX technique, was used in a capture assay. The aptamers bound to full length and PK treated PrPSc from hamster scrapie, as well as from sporadic CJD, vCJD, mouse scrapie, sheep scrapie, and white-tailed deer derived CWD. Binding was not observed to PrPSc from mule deer CWD, or from BSE, making these reagents among the first to show species or strain specificity. Strikingly, these aptamers were able to distinguish buffy coat samples derived from scrapie afflicted sheep from those of healthy animals in an electrophoretic mobility shift assay with 96% sensitivity and 100% specificity. These peptides and aptamers represent novel reagents with potential to open new avenues in prion disease research, surveillance, and therapeutics.