Poster 168 - Comparative analysis of the specific transcripts from Haementeria depressa leech

Poster 168. 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.527.

Comparative analysis of the specific transcripts from Haementeria depressa leech and Amblyomma cajennense tick salivary complexes by expressed sequence tags (ESTs) sampling

Faria, F.¹; Batista, I.F.C.¹; Junqueira-de-Azevedo, I.L.M.²; Ho, P.L.²; Sampaio, M.U.³; Chudzinski-Tavassi, A.M.¹

1 Laboratório de Bioquímica e Biofísica; 2 Centro de Biotecnologia, Instituto Butantan, São Paulo,SP, Brazil; 3 Departamento de Bioquímica, UNIFESP, São Paulo, SP, Brazil.

The saliva of blood-feeding animals usually is able to prevent the blood clotting during ingestion and/or assimilation, due to the presence of anticoagulant substances. A potent FXa inhibitor (lefaxin) and a fibrino(geno)lytic metalloprotease (hementerin) were purified from the salivary complexes of H. depressa leeches. Inhibitors of platelet aggregation, thrombin, and factor Xa from A. cajennense saliva have already been characterized. Aims: 1) construct plasmid cDNA libraries, from salivary complexes of H. depressa leech and A. cajennense tick; 2) sequence several clones of each library; 3) carry out a comparison analysis of gene-expression profiles using ESTs. The cDNAs were synthesized (Superscript plasmid system), selected by size, and inserted into pGEM11Zf+ plasmid, transformed in DH5a E. coli), and plated (2YT-amp agarose plates). Random clones were selected, their DNA extracted, and about 500 clones of A. cajennense library and 960 clones of H. depressa were sequenced. The set of expressed genes were obtained by assembling ESTs in clusters of unique genes and then analyzing their similarity against GenBank. In the H. depressa leech cDNA library, 898 independent clones were assembled (555 clusters: 102 contigs and 453 singlets) and important toxins involved in feeding were identified (about 10%) including serine proteases inhibitors, platelet aggregation inhibitors, antimicrobial, C type lectins, and metalloproteases. Among these 2,8% were characterized as candidates for lefaxin expression. From the A. cajennense tick cDNA library, 430 independent clones were sequenced (84 clusters). Among these, 9 codify serpins (2%), and one of these, showing high homology to a Factor Xa inhibitor, was elected to the expression.