Cloning And Expression Of Human Prion Protein Gene And Preparation Of The Antiserum

The open reading frame(ORF) of human prion protein gene (PRNP) was amplified by polymerase chain reaction (PCR) with the template of the total DNAs extracted from the human blood, which cloned into pMD18-T vector to construct the recombinant plasmid HOPRNP-T. By sequencing and analyzing the PRNP genes of the recombinant plasmid HOPRNP-T with DNAstar, it were indicated that the nucleotide and amino acid sequence of the PRNP gene exhibited 99.6% and 99.8% homology respectively with M13899 published in GenBank. The recombinant plasmid pET-homPrP was constructed by amplifying the segment of human mature prion protein gene(mPrP) with the template of plasmid HOPRNP-T and then linking to the expression vector pET30a(+) after digesting with enzymes. The pET-homPrP plasmid was transformed into E.coli BL21(DE3) and induced by IPTG for expression. The result of SDS-PAGE showed that the expression product was about 30ku of the fusion protein. The fusion protein was purified by Ni-NTA affinity chromatography after ultrasonic cleaving and it's concentration was 90%.The good immunoreaction of the fusion protein was detected by the Western-blot with mAb SAF70 as the antibody. As a immunogene and comparison immunogene, the purified fusion protein mPrP and E.coli BL21(DE3) contained pET30a vector were mixed to Freurds complete adjurent with the ratio of 1:1 respectively.8-week-old clean class BALB/c female mice were immunized by the way of hypodermic injecting the two kinds of immunogens with the dosage of 100ug/one.The two kinds of immunogens emulsified with the Freurds incomplete adjurent were injected to mice by the same way and dosage for strengthened immunity in the third and fifth week. After 3 days, the titer and specificity of anti-human mPrP serum obtained from eyes of mice immunized 3 times were detected by indirect ELISA and western-blot respectively. Results showed the titer of anti-human mPrP serum with strong specificity was 1:512. The experiment lay a foundation for researching the structure of human prion protein and preparing the corresponding monoclonal antibody.