RESUMO
We report the use of recombinant scorpion toxin in the form of fusion protein as antigen for mice immunisation. The aim is to produce protective antisera against lethal activity of the venom from Tunisian scorpion Buthus occitanus tunetanus, responsible for several annually reported human cases of scorpion stings. The gene encoding Bot III (the most toxic alpha toxin of Buthus occitanus tunetanus) was fused to the sequence encoding synthetic ZZ domains of staphylococcal protein A. The construct ZZ-Bot III was expressed in the periplasm of E. coli as a fusion protein and purified by affinity chromatography. The recombinant fusion protein was characterized and used as antigen to generate antibodies in mice. The antibodies against the recombinant protein neutralize the toxic venom (10 LD50/ml) and also confer protection for immunized mice against antigenically related mammal toxins.
Assuntos
Antivenenos/uso terapêutico , Modelos Animais de Doenças , Imunoterapia/métodos , Proteínas Recombinantes de Fusão/uso terapêutico , Picadas de Escorpião/terapia , Venenos de Escorpião , Animais , Antivenenos/farmacologia , Cromatografia de Afinidade , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Imunoterapia/normas , Camundongos , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/farmacologia , Picadas de Escorpião/etiologia , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/genética , Venenos de Escorpião/imunologia , Escorpiões , Proteína Estafilocócica A/genética , TunísiaRESUMO
Two toxin-like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross-reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins. In order to study structure-function relationships, their complete amino-acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three-dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion alpha-toxins specific to voltage dependent Na+ channels. The models showed also that amino-acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.
