| 1997 Nobel Prize in Physiology or Medicine was awarded to the American scientist Stanley B Prusiner for his discovery of an entirely new genre of disease-causing agents and the elucidation of the underlying principles of their mode of action. That agent is prion and the name is derived from the first letters of 'proteinaceous infectious particle', 'on' is added for the pronunciation convenience. Prions are infectious agents composed of proteins which differ from all other known pathogenic agents in that they lack a genome. They are responsible for transmissible spongiform encephalopathies (TSE), which affect both humans and animals. The prototypic prion disease found is scrapie. The epidemic of BSE in England and the newly found vCJD in human attracted more attention to prion diseases and the disease-causing proteins. There exist two prion protein forms, the pathological form, PrPsc (sc stands for scrapie), and normal cellular form, PrPc (c stands for cellular). These two proteins have the same amino acid sequence, the only difference lies in conformation. The propagation mechanism of the prion diseases is widely accepted that PrPsc transforms PrPc into the pathological form andmore and more PrPsc will gradually be produced and forms larger fibrillar aggregates. When a critical level of PrPsc has accumulated, the neurons die, resulting in large vacuoles. The affected brain regions become sponge-like in appearance, hence the name spongiform encephalopathies to describe all prion diseases.The facts that cellular prion protein is extensively distributed among tissues, its high similarity in different species and its indispensability in prion diseases development established its importance in the study of prion diseases. While the thorough knowledge of this normal protein will provide basis for understanding the disease mechanism and therapeutics. The studies about PrPc include its brain localization and putative physiological functions and the identification of its receptors (binding candidates). It's proved that PrPc functions through binding with another protein. So, the identification and characterization of its binding proteins will help to explore the physiological functions involved. To accomplish this goal, different methods were employed in this study.In the first part, the focus is to find the receptor molecules directly by screening two cDNA libraries with a recombinant construct PrPAP or as an alternative, to find an enriched area in the embryo brains and construct libraries from this brain region and perform the expression cloning as above. From PO cerebral cortex library, a positive pool was selected, but in the subscreening, the enrichment of the positive colony was not successful, so that no molecule was identified. Also in the embryo binding, there was no brain region which showed distinct staining. Instead, the whole brain was stained with the same intensity. When the approach to get a direct binding molecule was proved not fruitful, attention was turned to find binding sources for the purpose of further isolating the binding protein. The first consideredand also the relatively easier-obtained materials are some adhesion molecules. Results showed, only N-CAM 140 showed ambiguous binding. The other molecules (LI, CHL1, PO, TAG, F3, PrP) didn't show any binding. The next source proposed were glial cell lines since PrPc was proved to be abundant in myelin. Schwann and oligodendrocytes cell lines were tested for binding. Positive binding was found in both cell lines, but their binding percentages are not high enough to be considered as binding sources. Taking the heterogeneity of cell lines into account, primary cells were cultured for binding. Although hippocampal neurons and astrocytes also showed positive binding, cerebellar granule neurons are the more promising binding source because of their high yield, good binding and biological relevance.In the second part, PrP was constructed into a recombinant protein with human IgG Fc fragment (PrPFc) and used directly to interact with...
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