Establishment Of A Cell Line Expressing PrP/GFP Fusion Protein And Its Application In Prion Infection

Transmissible encephalopathies (TSE), also called prion diseases, such as Creutzfeldt-Jakob disease (CJD),Gerstmann-Strassler-Scheinker syndrome (GSS), kuru in humans, and scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in animals, are fatal neurodegenerative disorders with pathologies including neuronal cell loss,vacuolation, astrocytosis, and amyloid plaques . The conversion of cellular prion protein (PrP^C) into abnormal isoforms of prion protein (PrP^Sc) constitutes a fundamental feature of prion diseases . Prion protein (PrP) contains disulfide links (S–S), Asn-linked glycosylation sites (CHO),a signal peptide sequence (SP), an octapeptide repeat region(OR), a hydrophobic region (HR), and a glycosylphosphatidylinositol (GPI) anchor attached to its C terminus . One approach to studying TSE is the use of gene knockout mice and the derived cell lines, but now investigators pay more attention to gene knockout and transgenic cell lines.The biology of prion and the formation of PrP^Sc are still not well understood. Cell lines expressing PrP/GFP fusion protein provided useful tools to this approach. To generate this kind of cell lines , the eukaryotic expression plasmid pcDNA3.1(-) carrying PrP/GFP fusion cassette was used to transfect the Neuro-2a cells. Then this kind of cell lines can be used to take infected experiment .PCR primers were designed according to the published sequence of mouse PrP gene and the GFP sequence of pEGFP plasmid and amplified the sequence of mouse PrP1-34aa,PrP94-254aa,PrP112-254aa and GFP. The amplified DNA fragments were inserted into the pGEM-T vector separately to generate four recombinant plasmids, named pGEM-T-mPrP1-34aa,pGEM-T-mPrP94-254aa,pGEM-T-mPrP112-254aa and pGEM-T-GFP. Plasmi-d pGEM-T-mPrP1-34aa was digested with XbaI and AgeI, plasmids pGEM-T-mPrP94-254aa and pGEM-T-mPrP112-254aa were digested with EcoRV和HindI-II , plasmid pGEM-T-GFP was digested with AgeI和EcoRV, plasmid pcDNA3.1(-) was digested with XbaI和HindIII. The recovered bands of mPrP-1-34aa,mPrP94-254aa,GFP,pcDNA3.1(-) and mPrP1-34aa,mPrP112-254aa,GFP,pcDNA3.1(-) were ligated with T4 ligase to produce a fusion expression plasmid, named pcDNA3.1(-)-mPG94 and pcDNA3.1(-)-mPG112. We inserted the GFP bands after codons 94 and 112 of the mouse PrP gene respectively. After we got the cell line with PrP/GFP fusion protein. Then the two recombinant plasmids were used to transfect the Neuro-2a cells under the selection pressure of G418 antibiotics. The cells were subject to 30 passages under the selection and checked for the expression of PrP/GFP fusion protein by fluorescence and western-Blotting. The final cell lin-e was named as mPG94Neuro-2a and mPG112Neuro-2a. We infected the cells with scrapie infected-mice brain homogenates and we checked the infectivity of the cells with Western-Blotting every passing two times ten days later.After twenty days , we can checked PrPSc in the cell lines ,but not in Neuro-2a cell lines .So we can say that the cell lines with PrP(3F4)prion incubation times and supported prion replication .Also it is a useful tool for infected experiments .