Production and characterization of a polyclonal antibody against the bovine prion-like doppel protein

PRODUCTION AND CHARACTERIZATION OF A POLYCLONAL ANTIBODY AGAINST THE BOVINE PRION-LIKE DOPPEL PROTEIN Marco Rondena, Fabrizio Ceciliani, Chiara Bazzocchi, Saverio Paltrinieri Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria – Milano AIM OF THE STUDY: Doppel (Dpl) is a prion-like protein recently identified in mammalian. The sequence is notably homologous to that of PrP. Dpl is associated to neurodegeneration when ectopically expressed in transgenic mouse. The aim of the study is to raise and characterize a polyclonal antibody (pAb) against bovine Dpl to study the tissue distribution of this protein. MATERIAL AND METHODS. A synthetic peptide spanning from residues 67 to 81 of bovine Dpl sequence was synthetized. Using this peptide, a pAb (Dpl 67-81), was raised in rabbit and purified by means of Protein A affinity chromatography. In order to test the specificity of Dpl 67-81, bovine Dpl was cloned and overexpressed in E.coli in the expression vector PQE30 and purified following methal chelate affinity and bovine PrP was purified by means of cation-exchange chromatography and cobalt-chelate affinity chromatography. Homogenate of tissues were tested for the presence of Dpl after SDS-PAGE and Western Blotting. Blots were visualized using ECL. Immunohistochemistry on cryostatic sections using the avidin-biotin complex methods was performed. RESULTS: Dpl 67-81 strongly react against rDpl (titre =1:20000) and not against bovine PrP. Dpl was detected in testis, spleen, limphnodes by both immunoblotting and immunohistochemistry. In particular endothelial and stromal cells showed a granular cytoplasmic positivity. CONCLUSIONS: Dpl 67-81 can detect the expression of Dpl in tissues using both Western Blotting and immunohistochemistry. Moreover, it does not crossreact with PrP, and therefore it could be also used in experiments of functional blocking of Dpl in order to study its function and its relationship with PrP.