Establishment Of Immunohistochemistry Assay For Scrapie Detection And Characteristics Research For Mab 2H3 Which Identify Ovine PrP From Bovine PrP

The detection of scrapie is authorized by immunological assay mediated by monoclonal antibody, including ELISA, western blotting and immunohistochemistry (IHC) assay, in addition to histopathological test on brain. By the expression of Chinese short-tailed Han sheep PrP27-30 in E. coli BL21 (DE3) and preparation for anti-PrP27-30 of Chinese Short-tailed Han sheep monoclonal antibodies, the binding site and immunological characterization of the mAbs specific to ovine scrapie were systematicly studied in the present research. Two of the suitable mAbs, named 4C6 and 5F11, were selected to establish the method of immunohistochemistry assay. The total of 4922 samples from 26 provinces and autonomous regions of all over China were demonstrated for Scrapie. Meanwhile, using technique of gene mutation on the binding site of mAb 2H3, we discovered that I/M polymorphism of ovine PrP208 could identify ovine prion protein to bovine prion protein.Prnp gene of Chinese Short-tailed Han sheep was amplified from the Genomic DNA by the polymerase chain reaction and inserted into plasmid pET32a using T4 DNA ligase after digested with restriction enzyme, EcoRâ… and Hindâ…¢. The recombinant plasmid, named PrP-pET32a, was transferred to E. coli BL21 (DE3). The recombinant Chinese Short-tailed Han sheep PrP27-30 fusion protein, approximately 35kD, was obtained after 4h induced by IPTG. PrP null mice were injected subcutaneously with purified PrP27-30 fusion protein. After two times boosted, SP2/0 cell line and lymphocytes of the immunized PrP null mice were fused by means of lymphocyte hybridoma technique. Six hybridoma cell lines, which stably secreting monoclonal antibodies specific to PrP27-30 of Chinese Short-tailed Han sheep, were selected after three times of subclone. To find out the binding sites of mAbs 2H3, 4C6, 5Fll, 7F1 and 7F11, overlapping recombinant PrP27-30 which interval 15 amino acids every two consecutive peptides (excluding PrP peptide 2) were expressed in E. coli BL21 (DE3) and named PrP-peptide 1, PrP-peptide 2, PrP-peptide 3, PrP-peptide 4, PrP-peptide 5 and PrP-peptide 6 respectively. Binding sites of scrapie monoclonal antibodies were identified by Western blotting. Binding sites of five monoclonal antibodies specific to ovine scrapie are: 2H3 is between 199aa-213aa, 4C6 is between 153aa-154aa, 5F11 is between 154aa-168aa, 7F1 is between 214aa-227aa and 7F11 is between 154aa-168aa, respectively.ELISA characterization, western blotting characterization, IHC characterization of PrP^c and PrP^Sc between bovine and ovine were identified and analysized by mAbs 2H3, 4C6, 5F11 and 7F11 respectively in present research. The result showed that, 2H3 only reacted with ovine PrP but not bovine PrP; 4C6 strongly reacted with both bovine and ovine PrP; 5F11 and 7F11 gave the similar characterization which reacted more strongly with ovine PrP than bovine PrP.Acording to immunological characterization of Scrapie mAb 2H3, 2H3 binding site was compared to the other prion protein sequences published in Genebank by BLAST on NCBI web. The result showed that ovine PrP208, Isoleucine (I)/Methionine (M), owned polymorphism. By recombinant DNA technology, mutant fusion protein Mu-PrP-peptide5 which I replaced by M in 208 site was obtained. Western blotting showed that 2H3 only reacted with PrP-peptide5, but not Mu-PrP-peptide5. It implied that I/M polymorphism of ovine PrP208 could identify ovine prion protein to bovine prion protein.Two of the suitable mAb, 4C6 and 5F11, were selected to establish the method of immunohistochemistry assay. The total of 4922 samples from 26 provinces and autonomous regions of all over China were demonstrated for Scrapie during 2005 to 2006. All of the brain samples were scrapie negative.