The Subclone And Expression Mouse Prnp Gene, Mutain Construction And Characteristics Research Of The Expressions

Transmissible spongiform encephalopathies(TSEs), or Prion diseases, are chronic, fatal, infectious, neurodegenerative disorders occurring in both humans and animals.The TSEs pathogen which is called prion (proteineaceous infectious particle)or PrPSc (prion isform of scrapie)is the conformational conversion form of PrPC(Cellular form of Prion Protein).PrP is encoded by Prnp gene whose biological functions are still not clear.Compared to bovine and sheep, Mice are mostly used as an amimal model for TSE study considering its easy to operation,low raise costs,short incubation period .In this study,we cloned and expressed the core and mature sequence of the mouse Prnp,and then construted a PrP strain harbors a mutation(Pro→Leu).At last we make part characteristics initial research of the recombinant protein.1. Cloning and expression of mouse PrP core and mature sequenceWe make primers based on the Prnp gene sequence on the Gene Bank and than amplificated mice Prnp core and mature gene sequence with the plate of pMD18-T vector which contain the whole Prnp gene sequence.After getting the correct sequences ,we cloned them on pET-32a(+) and pET-28a(+) vectors ,transforred the recombinant vectors into BL21(DE3) and induced with IPTG. Purified the production by affinity chromatography.It is shown that the recombinant protein molecular weight is correct by SDS-PAGE.Western-blotting demonstrated that the aim sequence was correctly expressed and has good antigenicity. 2. The construction and prokaryotic expression of mouse Prnp core sequence mutation strainThe Prnp gene polymorphisms and diversity of mutation contiibute to PrP pathogenic.The mutation of PrP 101(Pro→Leu) can enhance mice morbidity of TSE,which analogous to the human prion protein mutation that cause Gerstmann-Str?ussler-Scheinker syndrome.So we mutate this site on pMD18-T-cPrP which constructed in research one by primer introduce mutated site.Sequence analysis are confirm correctly mutation and than cloned and expressed as conventional method.SDS-PAGE and western-blotting confirmed that we gained mice core Prnp gene mutation strain(P101L) successfully and its expression has good antigenicity.3. Characterisation of the mice recombinant PrPsSix recombinant mice PrPs were characterized.First,we conducted Matrix-assisted laser desorption/ionization spectra(MALDIS) analysis of the recombinant PrPs.The results showed that fusion protein can highly impact the exogenous conformation and characteristics.Second,we detected the protease-resistance of the inclusion protein and refolding protein ,both showed no protease-resistance.Finally,We tested the ELISA reaction of the six proteins to four McAbs against different epitopes.We find that PrP139～168aa is dominant epitope in all recombinant PrPs while PrP 109～112aa showed no reactivity as presumptively hiding in the tertiary structure.The above approaches provided the basic information for further study of TSE in nice model.