Cloning and prokaryotic expression of human prion gene

The open reading frame (ORF) of prion protein gene,Prnp,was amplified by PCR using total DNA extracted from human blood as template. The PCR product was ligated with pMD-18T vector and then transformed into E. coli JM109. The recombinant plasmid was sequenced and analyzed with Blast software. It shared 99.6 % homology at their nucleotide level and 99.8 % identity at deduced amino acid level with the corresponding sequences deposited in Genbank. The gene encoding mature human prion protein was cloned into pET-30a(+) vector and then transformed by electroporation into E. coli BL21(DE3) competent cells. The expected protein was expressed in E. coli BL21(DE3) harboring the recombinant plasmid pET-homPrP induced by 1.0 mmol/L IPTG at 37 ℃. The expressed fusion protein was purified by Ni—NTA affinity resin according to the manufacture's directions. SDS—PAGE analysis showed that the fusion protein accounted for 20 %-25 % of the total bacterial proteins and accumulated as inclusion bodies. Western blotting demonstrated that the fusion protein could be recognized by the anti-PrP monoclonal antibody and used as antigen for preparing the monoclonal antibody against PrP.