RESUMEN
In this work, the cDNA encoding a novel P-II type metalloproteinase from Bothrops asper venom glands was cloned, sequenced and used for DNA immunization of animals with accelerated DNA-coated tungsten microparticles and the helius Gene Gun system. Specific antibodies against B. asper venom antigens were induced in mice co-immunized with the plasmid encoding the P-II metalloproteinase together with an expression plasmid encoding the murine IL-2. Similarly, specific antibodies against B. asper venom antigens were also induced in a horse co-immunized with the plasmid encoding the P-II metalloproteinase, together with a plasmid encoding the equine IL-6. The equine antibodies induced by immunization with the P-II metalloproteinase encoding plasmid cross react with several proteins of B. asper, Crotalus durissus durissus, and Lachesis stenophrys venoms in western blot, demonstrating antigenic similarity between the cloned metalloproteinase and other metalloproteinases present in these venoms. Furthermore, the equine antibodies induced by immunization with the P-II metalloproteinase encoding plasmid completely neutralized the hemorrhagic activity of the whole B. asper venom and partially the hemorrhagic activity of C. durissus durissus venom. The neutralizing ability of the produced antibodies raises, for the first time, the possibility of developing therapeutic antivenoms in horses by DNA immunization using tungsten microparticles.
Asunto(s)
Antídotos/farmacología , Antivenenos/farmacología , Bothrops/inmunología , Venenos de Crotálidos/inmunología , Hemorragia/prevención & control , Metaloproteasas/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Hemorragia/inmunología , Caballos , Metaloproteasas/genética , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Plásmidos/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Vacunas de ADN/inmunologíaRESUMEN
Zinc-dependent metalloproteinases play a key role in the hemorrhage induced by viperid bite envenoming. In this work we report the cloning and sequencing of the cDNA from a novel P-III type metalloproteinase from Crotalus durissus durissus venom glands. The recombinant plasmid was used for DNA immunization in mice using accelerate DNA-coated microparticles with the Gene Gun system. The results showed that there is no significant difference in the efficiency of the immunization in mice when gold or tungsten microparticles were used. A pool of the sera from mice immunized with the metalloproteinase encoding DNA neutralized the hemorrhagic activity of C. d. durissus venom. The co-immunization with DNA encoding the metalloproteinase and a plasmid encoding the murine IL-2 increased the number of mice which show a specific antibody response towards C. d. durissus venom antigens. The neutralizing ability of the produced antibodies demonstrates that DNA immunization with tungsten microparticles may be used in strategies for antivenom production.