CLONING OF A SERINE PROTEINASE cDNA FROM Bothrops moojeni (CAISSACA) SNAKE VENOM

Vitorino, A.F.C.(1), Homsi-Brandeburgo, M.I.(1), Brites, V.L.C.(2), Bauab, F.(3), Selistre-de-Araújo, H.S.(4)

(1)Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia (UFU), MG, (2)Instituto de Biologia, UFU, (3)Faculdade de Medicina de Catanduva, SP, (4)Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, SP, Brazil.

Serine proteinases may represent about 20% of total snake venom protein composition. Acting on the victim’s blood coagulation cascade, these enzymes are not lethal, but they potentialize the toxic effects derived from envenomation. Because of these characteristics, they became important tools in the study of coagulation mechanisms and in the development of diagnostic tests, being models to synthesize therapeutic agents. In this work, we have cloned the cDNA encoding a previously isolated serine proteinase (BthT1) from Bothrops moojeni (Viperidae, Crotalinae) venom. The total RNA template of RT-PCR was extracted from the frozen venom gland of an adult female snake with TRIzoL reagent (GIBCO-BRL). Primers were designed using the Multalign software, based on the known serine proteinase N-terminal sequence and the 3’UTR region of homologue proteins. The amplification product was sequenced, cloned into the pGEMT Easy plasmid (Promega), and used to transform competent cells of E. coli DH5astrain. Restriction analysis of ampicilin-resistent transformed colonies revealed the expected dimensions for the vector (1015 bp) and insert (756 bp). The alignment of translated sequences of the RT-PCR product and the insert evidenced high homology. Blast analysis in the GenBank showed that the cloned sequence shared identity with snake venom serine proteinases like batroxobin (92%) and bothrombin (86%), conserving amino acid residues characteristics of the group. Additionally, some internal peptides of the serine proteinase cleaved with Cyanogen Bromide and analyzed by HPLC will be sequenced, in order to confirm your its correspondence to the cloned cDNA.

Supported by: CAPES and FAPESP. 

CORRESPONDENCE TO:

Ana Flávia Vitorino Cardoso, Avenida Brasil, 3460, Uberlândia, MG, CEP: 38.400-718, Brasil, Email: hannavitorino@hotmail.com