Isolamento e caracterização de peptídeos sintéticos em phage display pela interação com anticorpos policlonais anti-neuwiedase da peçonha bruta de Bothrops neuwiedi pauloensis

Abstract

Neuwiedase (Neu) is a weakly bleeding metalloproteinase, component of the Bothrops neuwiedi pauloensis venom. On the present work, it was used polyclonal antibodies against Neu, capable of neutralizing the bleeding effects of the brute venom of Bothrops neuwiedi pauloensis. These antibodies were used to select immonureactive peptides by the “Phage Display” technology. This technique basically consists in many peptide selection cycles (“Biopanning”) of 12 aminoacid synthesized peptides from a library, expressed on the surface of bacteriophage viral capsides. After three “Biopanning” cycles, 86 phage clones were obtained, 53 of them were sequenced, resulting in 48 different peptide sequences. On total, 40 of these sequences presented positive immunoreactivity through the “Colony Blotting” assay, from where it was used the anti-Neuwiedase policlonal antibodies. Probably, it was not possible to determine all the toxin epitopes, however, the Neu 40NTVNGFFRSMN51 site was the place where there was the highest alignment numbers, indicating this site as a probable epitope. The NLGM and KYN motifs present on Neu and on the Bn13 and Bn22 clones, respectively, also occur on the non-bleeding metaloproteinases LHF-II, fibrolase e lebetase on the venom of Lachesis muta muta and ACLPREF of Agkistrodon contortrix laticinctus, Adamalysina-ll of Crotalus adamanteus, Atroxase, Atrolysina-C e Ht-b of C. atrox. These motifs are also present on the bleeding metaloproteinases BOJUMET I, BOJUMET II and BOJUMET III of Bothrops jararacuçu, besides the protrombine activating toxine Berythractivase of Bothrops erythromelas. The Bap 1 toxin of Bothrops asper has the TKYN and NXXFRS motifs, in common with Neu and the Bn 22 e Bn 13 peptides, respectively. These selected peptides can present a potential use on the antivenoms production, or even as vaccinal antigens. To this use, these clones are going to be tested very soon to their immunogenicity in vivo.