NR AXOR
AU Horigan,V.; Saunders,G.C.; Tout,A.C.; Sauer,M.
TI Characterising TSE Strains by 2 Dimensional Immunoblotting
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Natural and Experimental Strains P02.33
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: Experimental transmissible spongiform encephalopathy (TSE) strains are defined by stable forms of prion that, upon inoculation in animals, cause disease with unique incubation time and lesion profile. Various distinct scrapie strains have been isolated, however, until recent identification of two atypical phenotypes, evidence indicated that BSE in cattle was caused by a single strain. Traditional methods used for strain discrimination have relied on measurements of disease characteristics in mouse models. Biochemical strain typing approaches are largely based on the molecular characterisation of PrPsc using 1D immunoblotting (IB). This approach has limitations however and 2D IB offers much promise for identifying strain specific differences in PrPsc which 1D IB can not readily resolve.
Aims: To characterise PrPsc from mice infected with experimental TSEs by 2D IB with a view to distinguishing between ME7, 22A, 87V , 79A and 301V experimental strains and to investigate the potential to identify distinct TSE strains in sheep (natural and experimental disease).
Methods: 10% brain homogenates were analysed by 2D IB using 6H4 and P4 antibodies. Deglycosylation was carried out using PNGase F enzyme in an overnight incubation at 37oC.
Results: Experimental TSEs in mice models exhibited strain specific changes within the total PrP and PrPsc profiles. Analysis of natural and experimental TSE strains in sheep showed differences between the 2D profiles after deglycosylation, with ovine BSE and CH1641 cases exhibiting fewer PrPres isoforms than ovine scrapie and SSBP-1. A greater range of basic glycosylated PrPres isoforms were also detected in SSBP-1 than CH1641 profiles, indicative of a strain specific PK cleavage site in both cases.
Conclusion: These studies indicated that the high power of 2D IB to resolve PrP glycoforms and truncated products provides the possibility of distinguishing subtle multiple differences between strains which is not readily possible with 1D methods.
This work was funded by Defra, UK
AD V. Horigan, G.C. Saunders, A.C. Tout, M. Sauer, Veterinary Laboratories Agency, Molecular Pathogenesis and Genetics Department, UK
SP englisch
PO Schottland