Researching For The Interaction Between The Protein Of PrP And VCP By The Technique Of Yeast Two-hybrid And RNAi

Transmissible spongiform encephalopathy(TSE) is caused by the prions a neurological disease. But the development of the disease occurred and has not been given to solve, about prions cause of all kinds of disease pathogenesis mechanism have not been clearly explained, so prions caught the attention of researchers all over the world. There are two types of prion usually structure form, one is not the normal form of the pathogenicity of the PrPc, another kind is the normal PrPc into a pathogenic form of PrPsc. The present study think normal form of PrPc in certain conditions error can be folded into a disease-causing form PrPsc, so PrPc PrPsc space configuration to the change of mad cow disease pathogenesis is the key. In the process of change configuration, and PrPc and PrPsc interaction has the important meaning of the protein.This experiment is constructed pMyr-VCP (1-891), pMyr-VCP (799-1560), pMyr-VCP (1447-2406), three of the material, through the double enzyme cut identification, sequencing identification, and prove the bait plasmid and hunting material construction of success; The lab has been successful before constructing the bait plasmid pSos-PrP (23-231), we will bait plasmid, hunting material grains and control plasmid co-transformation yeast cde25H a, detection products in its expression are yeast cells from activation function and orientation of the cell membranes; And will bait plasmid and hunting material grain co-transformation cdc25Ha yeast feeling state, for their experiments, the results show that pSos-PrP (23-231) and pMyr-VCP (1447-2406) of expression fusion protein can interact, again through the immune were further validation yeast double hybrid precipitation of the results, and the prion physiological function and mechanism of error PrP fold to do further research.In addition, this experiment using a technique called RNA interference to VCP PrPc into a research PrPsc influence. To VCP gene design four interference targets, and the success of VCP gene construction four interference plasmid, respectively pGPU6-GFP-VCP1-shRNA, pGPU6-GFP-VCP2-shRNA, pGPU6-GFP-VCP3-shRNA, pGPU6-GFP-VCP4-shRNA, and through the quantitative Western Blot out the best effect of interference detection plasmid is pGPU6-GFP-VCP4-shRNA. Will the plasmid ZhuanRan to already infected nerve tumour cells, the Western Blot test PrPsc change, when VCP reduce the amount of protein expression, to the change of PrPsc PrPc is relatively less. In animal experiments, has three set of normal mice infected wild type, PrPsc mice, PrP knockout mice, PrPsc quantity to have differences exist in the case, testing mice VCP protein expression quantity, and found that, PrPsc mice infected with the highest content of VCP. So VCP PrPc to PrPsc force-induced conformation transition of to have the function of promoting.In this essay,the recombiant 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 pMyr-VCP(1-891) pMyr-VCP(799-1560)、pMyr-VCP(1447-2406) 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 pSos-PrP(23-231) and prey plasmids were transformed into yeast cdc25Ha competence to carry out each experiment,the results of the analysis to prove, pSos-PrP(23-231) and pMyr-VCP(1447-2406) expressed the fusion protein can interact, and then through further verif immunoprecipitation of yeast two-hybrid results of the prion protein PrP occurred in the physiological function and mechanism of misfolding further study.