Expression And Purification Of Mouse PrP-like Protein Shadoo Preparation And Identification Of Polyclonal Antibody Against Shadoo

PrPsc (Scrapie Prion Protein)is the main causative agent of Transmissiblengiform 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.On account of the development of bioinformatics, by comparative genomics we have found a new membrane protein named Shadoo protein in gene database, which has the same construction as prion protein, and SPRN, the gene sequence of prion protein is always conservative from fish to mamMal.At present, we have not found monoclonal antibody in mice aiming at Shadoo protein globally, in addition, we can't obtain it unless we get the SPRN gene knocked-out mice stock. The process is time-consuming and needs lots of effort which influence empirical study pacing seriously. So preparing polyclonal antibody with fine reactivity and obvious specificity is becoming a necessary condition for deep research of Shadoo protein.In this experiment, a pair of specificity primer is designed aiming at SPRN. With the whole blood genomic DNA being as formwork, we get complete open reading frame of SPRN through PCR amplification.The product in it will be digested by incision enzyme (BamH I and Hind III) introduced in when we design the primer. After gelatum, reclamation and purification, the SPRN gene fragment will be directly inserted into procaryon expression vector which has already been digested and enzyme digestion, then we can obtain positive clones through identification of enzyme digestion. Recombinant plasmid pET-32a-SPRN was transformed into host bacterium Rosetta-gami(DE3), then expressed inclusion body proteins were collected after treated with 0.4 mM IPTG at 30℃to stay overnight.Expressed Shadoo protein conducted SDS-PAGE analysis, and a specific band was found at 33Ku,via Western-blot detection,expressed protein could be recognized by HIS tag antibody. It was conducted affinity chromatograph to purify interest protein via 6His tag that located at N-terminal of it,and then purified inclusion body protein was conducted dialysis renaturation through glutathione oxidation-reduction system, purification concentration to 735 ug/ml.Purified Shadoo protein was injected into musculi coll and multipoints of hypo-skin to imMune New-Zealand albino rabbit with a concentration of 0.5 mg per rabbit,afer being imMuned 5 times, antibody valence of Shadoo protein serum was evaluated at 1:12 800 with the method of indiect ELISA.With the obtained Shadoo polyclonal antibody to detect endogenous Shadoo protein in mouse brain and exogenous Shadoo protein that expressed in cells after transfection,the results showed that a specific expressed band was found at 20 Ku.Shadoo protein obtained by prokaryotic expression possessed comparatively srong imMunocompetence. Polyclonal antibody that anti-Shadoo protein was obtained successfully,and possessed comparatively high valency and specificity.It also possessed very fine imMunogenicity through Western-blot detection.