| Autor: |
Silva CJ; Produce Safety and Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, California 94710, United States of America., Cassmann ED; Virus and Prion Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010, United States of America., Greenlee JJ; Virus and Prion Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010, United States of America., Erickson-Beltran ML; Produce Safety and Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, California 94710, United States of America., Requena JR; CIMUS Biomedical Research Institute & Department of Medical Sciences, University of Santiago de Compostela-IDIS, 15782 Santiago de Compostela, Spain. |
| Abstrakt: |
In sheep, the transmissibility and progression of scrapie, a sheep prion (PrP Sc ) disease, is strongly dependent upon specific amino acid polymorphisms in the natively expressed prion protein (PrP C ). Sheep expressing PrP C with lysine (K) polymorphism at position 171 (K171) are partially resistant to oronasal dosing of classical sheep scrapie. In addition, scrapie infected sheep expressing the K171 polymorphism show a longer incubation period compared to sheep homozygous (glutamine (Q)) at position 171. Quantitating the amount of the K171 polymorphism in a sheep scrapie sample can provide important information on the composition of PrP Sc . A tryptic peptide, 159 R.YPNQVYYRPVDK.Y 172 , derived from the digestion of 171K recombinant PrP, was identified as an analyte peptide suitable for a multiple reaction monitoring-based analysis. This method, using 15 N-labeled analogs and another internal peptide from the proteinase K-resistant core, permits the simultaneous quantitation of the total amount of PrP and the proportion of K171 polymorphism in the sample. Background molecules with similar retention times and transitions were present in samples from scrapie-infected sheep. Proteinase K digestion followed by ultracentrifugation-based isolation or phosphotungstic acid-based isolation were employed to minimize the contribution of those background molecules, making this approach suitable for quantitating the amount of the K171 polymorphism in heterozygous scrapie infected sheep. |
