Development Of Sandwich ELISA For The Detection Of Prion Protein In Sika Deer

Chronic wasting disease（CWD）, a unique prion disease that affects elk anddeer, Known historic distribution of chronic wasting disease in farmed andfree-ranging cervids in North America. The mule deer,white-tailed deer, and RockyMountain elk all can be infected with CWD. Features of CWD in clinical stages arenot obvious. When animals in terminal clinical stages are weight loss and behavioralchanges. Chronic wasting disease(CWD) is designated prion disease because of theirassociation with aberrantly refolded isoforms of the prion protein, a normal cellularglycoprotein. Because the space structure changes, the character of the prion proteinwas changed. Prion has the ability of anti-protease digestion to leave some fragments.It is the basis for the immunological detection of prion disease.We extracted the genome of Sika deer, and used the specific primers to amplifythe core fragment of the prion protein. When the vectors with the restriction enzymeof BamHⅠand HindⅢ digestion, and connect with the target gene to constructrecombinant expression vectors: pET-PrP-His and pET-PrP-Trx. After inducedexpression many times, we get two recombinant proteins: PrP-His and PrP-Trx. Weuse the PrP-His combined with the commercialization Mab SAF31and HRP labeledantibody to establishment an indirect ELISA. We used PrP-Trx immunize Balb/Cmice to produce antibody, successfully obtained a hybridoma cell line “3C6”. Weused PrP-His immunize rabbit to produce antibody, when antiserum purified byacid-ammonium sulfate, used commercialization HRP to label. With monoclonalantibody3C6and HRP label antibody to build a double-antibody sandwich ELISAmethod, repeatedly optimized experimental conditions. We collect120brains of Sikadeer to actual sample test, the experimental results of the double-antibody sandwich ELISA are consistent with the other detection methods,concidence rate was96.7%(116/120).