Researching For The Interaction Between The Protein Of PrP And Shadoo In Yeast Two-hybrid System

Transmissible spongiform encephalopathy (TSE) is a human and animal's central nervous system degenerative disease caused by the Scrapie Prion Proein (PrPsc) which is the isoform of the Celluar Prion Protein (PrPc). Several TSEs have been reported, such as Creutzfeldt-Jakob disease (CJD), Kuru disease (Kuru), Transmissible mink encephalopathy (TME), Cervine chronicity wasting disease (CWD), Bovine spongiform encephalopathy (BSE).The typical clinical symptoms of TSE include progressive ataxia, thrill, unsteadiness, dementia, perceptual hyperesthesia, abnormal behavior. The typical feature is the acumulation of PrPsc which is abound with theβ-fold and possesses partial protease resistance according to the histopathology. PrPsc is from the PrPc that is abound with a-Helix, which mechanism is still unknown. Some researchers presume that there may be some other proteins affect in the pathogenesy of TSE, but these proteins have not been defined. So searching for the proteins interacted with PrPc is significant for the research of the transform mechanism, and also the physiology function of PrPc.There are several large scale and high-flux technologies can be used to study protein-protein interaction, such as protein chip, biology mass spectra, bioinformatics analysis, yeast two-hybrid system. Yeast two-hybrid system has became one of the most drastic methods in the study of protein-protein interaction.Stratagene CytoTra^TM two-hybrid system is based upon generating fusion proteins whose interaction in the yeast cytoplasm activates the Ras-signaling pathway, inducing cell growth, so it is also called as SOS restorey system. Human Sos (hSos) protein anchored in the inboard cellular membrane can activate the Ras-signaling pathway, and cdc25 is the yeast homologue of the human Sos (hSos) gene. The CytoTra^TM two-hybrid system uses the yeast temperature-sensitive mutant strain cdc25H which contains a point mutation at amino acid residue 1328 of the cdc25 gene. The cdc25 mutation in the cdc25H strain prevents growth at 37℃, but allows normal growth at the permissive temperature (22℃-25℃). In the CytoTrap^TM two-hybrid system, expression of hSos and its subsequent localization to the plasma membrane allows the cdc25H yeast strain to grow at 37℃. All the interactions of protein-protein happen in cytoplasm, so it can overcome the limitations of the traditionary yeast two-hybrid system.In this essay,the recombiant bait vectors and prey vectors of yease two-hybrid system were obtained after verified by endonucleases digestion analysis and sequencing. Meanwhile, control co-transformation experiments were done using the pSos-PrP(23-216),pSos-PrP(23-198),pSos-PrP(23-192),pSos-PrP(23-178),pSos-PrP(23-163),pSos-PrP(23-159),pSos-PrP(23-153),pSos-PrP(23-142),pSos-PrP(23-130?,pSos-PrP(23-126?,pSos-PrP(23-108),pSos-PrP(23-90) and each prey plasmid were co-transformed into cdc25Ha, detecting their expression product in yeast cells whether or not self-activation and the positioning of the situation on the cell membrane; the bait plasmids and prey plasmids were transformed into yeast cdc25Ha competence to carry out each experiment, the results of the analysis to prove, pSos-PrP(23-126) and pMyr-SHo(25-77) expressed the fusion protein can interact, and then through further verify inununoprecipitation of yeast two-hybrid results of the prion protein PrP occurred in the physiological function and mechanism of misfolding further study.