Study On Detection Of Prion Protein Based On Surface Plasmon Resonance Technology

PrPSc (Scrapie Prion Protein) is the main causative agent of Transmissible Spongiform Encephalopathy (TSE). Prion Protein has two conformation forms,a normal form- PrPC (Cellular Prion Protein), and the other abnormal form– PrPSc. As the common view, PrPSc is thought to be converted from PrPC, which can trigger prions disease. The main different performance between PrPC and PrPSc is the resistance to the proteinase K digestion. Both of them can response to the antibody, but after proteinase K digestion, normal PrPC will be fully digested, and the abnormal PrPSc only partially digested, still retained section of 27-30 ku fragments which could be identified by antibody. It provides a theoretical basis for the establishment of the normal PrPc detection method.At present,more effective detection method is based on the response between antigen and immune antibody, capillary electrophoresis methods and some method of the enzyme tinked assay, fluorescence scanning methods. As far as concerned resently study, detection of prion protein by Surface Plasmon Resonance has not been reported. It has the advantage of real-time, label-free, high sensitivity, specificity, reproducibility, and no radioactive quantitative measurement and automation, and is fit for high-flux samples screening of TSE. In this experiment, murine prion protein expressed in Escherichia coli was purified for the establishment of Surface Plasmon Resonance method to detecte PrPC, which made a foundation on the development of PrPScdetection method.The 624bp fragment of the PRNP coded PrPC was amplified by PCR method which the template of total DNA extracted from murine livers. The purified target gene fragment was cloned into the vector termed pMD18-T Simple. After sequencing the positive clons, the PRNP gene digested by BamH I and HindⅢwas subcloned into the expression vector pRSET-B digested by the same two enzymes. After the identification, the positive recombinant plasmid pRSET-B -PRNP was picked up.The recombinant plasmid pRSET-B -PRNP was transformed to the host E.coli termed BL21(DE3),the recombinant protein was analysed by SDS-PAGE and the specific expression band was detected by Western-Blotting assay with the special monoclonal antibodies HIS.The optimal reaction conditions of the Surface Plasmon Resonance method was established: 6H4 antibody is immobilized in 10 mM, pH4.5 sodium acetate; Regeneration buffer is glycine ,10 mM pH2.0. The standard curve in HBS buffer was obtained and regression equation y = 0.2614x + 0.1763 (R2=0.9993) was got by a regression analysis. The results indicated that 30 ng/mL of antigen was the lowest detectable limit of Surface Plasmon Resonance in HBS buffer. The standard curve in milk was obtained and regression equation y = 0.2614x + 0.1763 (R2=0.9996) was got by a regression analysis. The results indicated that 100 ng/mL of antigen was the lowest detectable limit of Surface Plasmon Resonance in milk.