Molecular Mechanism Of Prion Species Transmission Barriers Between Mouse And Hamster

OBJECTIVE: Prion diseases are a group of neurodegenerative diseases caused by misfolding of prion protein,also known as transmissible spongiform encephalopathy(TSE).The proportion of ?-helical structure of misfolding prion protein decreases,and the proportion of ?-sheet structure is obvious,which causes the accumulation of misfolding prion protein to become fibrill,causing apoptosis of nerve cells.This misfolding prion protein is called Pr PSc and can be transmitted to animals through food,blood and etc.,and the Pr PSc entering the host can induce misfolding of the host normal prion protein,thereby leading the infection of the disease.The transmission of prion proteins between mouse and hamster has species barrier effect,which shows that the latency of experimental animals is prolonged and the incidence is low.In addition to the cause of the strain,the transmission species barrier of prion proteins is mainly caused by the difference of amino acid sites of heterologous prion proteins.There are 11 different amino acid sites between mouse and hamster,and each site has different effect on the transmission of prion protein between mouse and hamster.On the basis of hamster prion protein,we construct multi-sites mutanted prion proteins with different mutant sites.Using PMCA technology and recombinant prion protein technology in vitro,we explored whether different chimeras could overcome the species barrier between mouse and hamster.METHODS: On the synthesized plasmid template,mutant primers were designed to mutate hamster prion amino sites into mouse's.After sequencing correctly,the mutant vector was transfected into E.coli BL21 to induce the expression of recombinant prion protein.The purified recombinant prion protein was inocubated with mouse Pr PScin the PMCA experiment to observe whether the product of PMCA has Proteinase K resistance through the digestion of Proteinase K.RESULTS: 7 recombinant prion protein genes with multiple sites mutations were successfully constructed,including four 3-mutant sites prion proteins,two 4-mutant sites prion proteins and one 5-mutant sites prion protein.After correct sequencing,the transfected E.coli was induced to express prion protein,and the prion protein was obtained by Ni-NTA affinity chromatography.After the preparation of the substrate,PMCA experiments were carried out with mouse Pr PSc added into substrate.Then the Werstern Blot test the Proteinase K resistance of Pr PSc conformation.The results showed that the transformed conformation of Ha Pr P(M111V-M138I-I204M)yielded17 k D fragments with pathogenicity,and other mutants had 14 k D fragments without pathogenicity after digestion.CONCLUSION: PMCA results showed that only HaPrP(M111V-M138I-I204M)mutant obtained the pathogenic conformation with 17 k D fragment digested by PK,and nonpathogenic 14 k D fragment was found.However,the other 3 multisites mutants and 4 multisites mutants and 5 multisites mutants obtained not only 14 k D fragments of nonpathogenic conformation after digested by PK but also 17 k D fragments of pathogenic conformation after digested by PK.The simultaneous existence of these two different conformations may prevent inoculated animals from getting sick and unable to cross the species barrier.At the same time,M111 V,M138I and I204 M were not completely consistent with the previously results that G54 ins,I138M and M204 I matants affected the transmission of prion proteins between mouse and hamster.It demonstrates that the effect of mutant site is not simple superposition effect,and it is possible that these sites also have spatial interactions.In this paper,through the recombinant prion protein and PMCA technology,three-mutants prion that overcame the species barriers between mouse and hamster was obtained,and we demonstrate the key amino acids for the propagation between mouse and hamster and the conformational selection theory is further verified.