BmCistatina, Inibidor de cisteinoproteases presente em corpo gorduroso de carrapato Boophilus microplus: Clonagem, expressão, purificação e caracterização

• Autor(a) principal: Lima, Cássia Arantes de [UNIFESP]
• Data de Publicação: 2006
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: http://repositorio.unifesp.br/handle/11600/9149
• topic: Boophilus microplus; hematófago; inibidores; cistatina; cisteinoproteases; estefinas

The tick economic importance is directly related to its blood feeding behavior. Among the species that belong to the Ixodidae family the Boophilus microplus tick has great importance in veterinary medicine. It is an ectoparasite responsible for disease transmissions such as: the babesiosis and fever of cattle. The tick control has been done by using acaricides, however alternative methods as the immunological control have been investigated. The protease inhibitors have an important role in the proteolysis regulation of endogenous or exogenous enzymes from all organisms. The protease inhibitors present in hematophagous animals are important molecules in the control of innumerous proteases involved in the host’s hemostatic and immune systems. The present work describes the characterization of a recombinant cysteine protease inhibitor, Bmcystatin from the fat body of B. microplus. The complete nucleotide sequence of the Bmcystatin (298 pb) was obtained by random sequencing of clones from a fat body cDNA library. The recombinant protein was produced using the E. coli BL21 SI expression vector pET26b. The Bmcystatin expression was induced with 0.3 M NaCl and the soluble protein extracted by French press. The Bmcystatin purification was performed by affinity chromatography on Ni-NTA agarose resin followed by ionic exchange chromatography on HiTrap Q column. Purified Bmcystatin presented molecular mass of 11 kDa and high inhibitory activity for cathepsin L with Ki value of 0.1 nM. The Bmcystatin amino acid sequence presented 70% similarity to an Ixodes scapularis tick cystatin. The expression analysis of Bmcystatin by semiquantitive RT-PCR showed DNA band amplification only in the fat body cDNA preparation. On the other hand, the protein recognition by anti-Bmcystatin antibody was observed in the fat body and salivary gland extracts of B. microplus by Western blot. In conclusion, this work generated the following tools for future studies: the anti-Bmcystatin antibody and the complete nucleotide sequence of Bmcystatin gene, which will be important to confirm Bmcystatin localization in B. microplus, and it will allow us to use the RNA interference methodology for functional studies, respectively.