Development of oligobodies (aptamers) against the bovine Prp (prion).

VALDIVIESO, A.G; TAMINELLI, G.L.; TEIBER, M.L; DANKERT, M.A.; SANTA COLOMA, T.A.

Rosario, Santa Fé, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

SAIB

Oligobodies (Obs) are difined as an oligonucleotide-based synthetic reagent that behaves as an antibody. They can be self-amplified by PCR, which increases the detection limits to the levels of PCR detection methods, something impossible to obtain with antibodies. The main objective of this work was to develop polyclonal Obs against synthetic peptides corresponding to different PrP cellular bovine (PrPC) regions and to test them in its ability to recognize PrPC and recombinant PrP (rPrP). Three synthetic peptides were designed for use in the selections. In order to select the Obs, different oligonucleotide libraries (between 55 and 75 to mer internal random sequence) were constructed whit consensus sequences in each end to be able to amplify by PCR the selected oligonucleotides. Selections were performed incubating membranes saturated with the peptide target and single strand library, were made washings and the oligonucleotides eluted were amplified by PCR with one of primers marked with biotin, obtaining themselves the polyclonal Obs for each one of different synthetic peptides. The Obs against different peptides was test in their ability to recognize PrPC bovine and rPrP proteins using a mixture of proteins on a Western blots. Alkaline streptavidine-phosphatase was used as develop system. As result the Obs polyclonal obtained were able to recognize rPrP (50 ng) in Western blot. Although his specificity were not optimal, tests could to be used directly for in alive if a step previous with proteinasa K is added, that destroys so much PrPC protein as the other protein, but does not affect infectious protein PrPSc. In addition, an alternative in the search of greater specificity and affinity, we will introduce mutations random on the Obs monoclonal whit low affinity using a synthesizer.