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Poster 54. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.413. |
Production, evaluation and characterization of recombinant sphingomyelinasesof the fiddlebackspiders L. boneti, L. reclusa and L. laeta.
1Ramos-Cerrillo B.; 1Olvera, A.; 1Stock, RP; 2Vázquez, H.; 1Odell, G.; 3Estévez, J.; 2Paniagua-Solís, J.; 4de Roodt, A. and 1Alagón A.
1 Instituto de Biotecnología-UNAM, Cuernavaca México.2 Laboratorios Silanes, México. 3 Instituto Bioclon, México. 4 INPB-ANLIS “Dr. Carlos G. Malbrán”, Buenos Aires, Argentina.
The bite of Loxosceles spiders can induce dermonecrotic lesions and systemic syndromes such as kidney failure and hemolysis. In Mexico, around 40 species of the genus Loxosceles spider are found. In recent studies it has been shown that a sphingomyelinase D (SMD) activity found in the venom is responsible for the pathological effects of the Loxoscelesspider bite. Even though the medical problem posed by loxoscelism has been recognized for some time, a polyvalent commercial and efficacy-tested antivenom is not available. In this study we generated recombinant SMD antigens for the production of a neutralizing antivenom against the toxic effects of SMD. For this purpose, total RNA was extracted from the venom glands of L. boneti, L. reclusa and L. laeta and cDNA was obtained by RT-PCR using primers complementary to the nucleotide sequences coding the first six amino acids of the mature proteins. The PCR products obtained were cloned and sequenced, establishing the complete bona fide sequences of the various SMDs. The amplified fragments were cloned in E. coli-based expression vectors and conditions for expression of active soluble SMDs were established. The final constructs encoded histidine-tagged polypeptides of 30-34 kDa. Amino- and carboxy-terminally tagged proteins were generated and their activities and immunnogeic properties compared. The recombinant proteins obtained had all the biological properties ascribed to the native mature toxins including the enzymatic and dermonecrotic activities. Using these recombinant proteins to immunize rabbits we were able to produce antibodies capable of conferring protection against the effects of the venom in mice, and evaluated the cross-neutralizing potential of the antibodies against SMDs of different origin.