Expression and Detection of Recombinant Sheep Prion Protein (RecShPrP^C) Using Human Neuroblastoma Cells and Western Blotting

Abstract

The objective of this study was to over express the sheep normal prion protein (RecShPrPc) in SK-N-SH cells using a mammalian expression vector with C-terminal 6XHis-tag for large scale purification by Nickel-Nitrilotriacetate column chromatography. The full length prion open reading frame was PCR amplified and the complete coding sequence (PrP25-234) including the signal peptide from both C and N terminal ends of the protein was subcloned into pcDNA6.0 plasmid. The orientation and integrity of the cloned PCR product in the mammalian expression vector was confirmed by DNA sequencing. The prion gene was transfected using Lipofectamine2000 reagent and successfully over expressed in SK-N-SH cells. The cells were lysed and the over expressed prion protein was detected using Western Blot. Production of RecShPrPC using this method would ensure the availability of large amounts of this protein for further downstream applications. In vitro production of the full length PrPC and its detection is important in further studies aimed at deciphering the biochemistry and structure of prion proteins.