XXXV Reunião Anual da SBBqResumoID:8439


Isolation and characterization of a neutrophil elastase inhibitor from B. microplus saliva similar to antimicrobial peptides


1Sasaki, S.D., 1Oliveira, G.P., 1Lima, C.A. and 1Tanaka, A.S.



1Departamento de Bioquímica, UNIFESP-EPM, 04044-020, São Paulo, SP, Brasil.

E-mail: ssasaki.bioq@epm.br


The hard tick Boophilus microplus infests bovine cattle causing heavy losses in meat, milk and leather productions. This ectoparasite is also the vector of bovine diseases as babesioses and anaplasmosis. The tick control is still a challenge for the cattle producers because of the high costs and less knowledge about tick physiology. For several years, our group has been studied serine proteinase inhibitors from different tissues and life stages of Boophilus microplus, among them neutrophil elastase inhibitors. In this work, an active elastase inhibitor was purified from B. microplus saliva (BmSEI) using classical chromatographic steps: two ion exchange chromatographies (HiTrap Q Sepharose FF and Resource Q columns) and a RP-HPLC chromatography on C8 column. Purified BmSEI showed to be a strong neutrophil elastase inhibitor with Ki= 2 nM. The N-terminal sequence of BmSEI after RP-HPLC determined by automatic amino acid sequencing showed similarity to  a N-terminal sequence of a predict protein from a gene obtained from a B. microplus cDNA fat body library (BmSEI gene). The protein sequence analysis presented similarity with two antimicrobial peptides described in: Boophilus microplus (microplusin) and Amblyomma hebreum (hebraein) by BLAST program. The analysis of the predict protein from BmSEI gene also showed that the mature protein molecular weight is 9.8 kDa, and a signal peptide was identified by analysis using SignalP 3.0 program. In an attempt to express the recombinant BmSEI, the gene was cloned into pPIC9 plasmid to use Pichia pastoris system. The recombinant BmSEI will be used to confirm if BmSEI is a molecule with two different activities: neutrophil elastase inhibition and antimicrobial activity. Supported by: FAPESP, Pronex-FAPERJ and CNPq.