RESUMO

A novel peptide named Pg8 was purified from the venom of the South African scorpion Parabuthus granulatus and its primary structure was determined. It contains 63 amino acid residues tightly folded by 4 disulfide bridges. The gene coding for this peptide was cloned from a cDNA library. By recursive PCR strategy a hybrid gene was constructed having a factor X recognition site for proteolysis and a modified sequence for preferential codon usage of E. coli. A pQE30 molecular vector already contained a His-tag was used for expression. This construction was expressed in BL21 and Origami strains. The fusion protein from inclusion bodies was separated by HPLC (yield approximately 5mg/L) and properly folded in vitro. Lethality tests showed that the recombinant peptide was toxic and was used to immunize mice. A volume of 0.25ml of the anti-serum produced was capable of protecting up to 3 LD(50) doses of pure toxin Pg8 but also, and more importantly, the entire soluble venom.

Assuntos

Antivenenos/genética , Venenos de Escorpião/genética , Venenos de Escorpião/imunologia , Escorpiões/genética , Sequência de Aminoácidos , Animais , Anticorpos/análise , Antivenenos/imunologia , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Genes/genética , Genes/imunologia , Camundongos , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Venenos de Escorpião/enzimologia