Navegando por Palavras-chave "Boophilus microplus"
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- ItemAcesso aberto (Open Access)BmCistatina, Inibidor de cisteinoproteases presente em corpo gorduroso de carrapato Boophilus microplus: Clonagem, expressão, purificação e caracterização(Universidade Federal de São Paulo (UNIFESP), 2006-12-31) Lima, Cássia Arantes de [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)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.
- ItemSomente MetadadadosBmeystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus(Elsevier B.V., 2006-08-18) Lima, Cassia A.; Sasaki, Sergio D.; Tanaka, Aparecida S.; Universidade Federal de São Paulo (UNIFESP)The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type I cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). the Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M-r of I I kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-aga-rose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of I I kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C I cysteine peptidase inhibitor with K-i value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. the rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. the present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis. (c) 2006 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosBmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae(Elsevier B.V., 2008-02-01) Sasaki, Sergio D. [UNIFESP]; Lima, Cdssia A. de [UNIFESP]; Lovato, Diogo V. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Torquato, Ricardo J. S. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. the inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271 Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (K-i 0.4 and 0.3 nM) and subtilisin A (K-i 1.4 nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (K-i 50 nM) and BmSI-6 (K-i 2.2 nM). the BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi. (C) 2007 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosBmTI antigens induce a bovine protective immune response against Boophilus microplus tick(Elsevier B.V., 2002-03-01) Andreotti, R.; Gomes, A.; Malavazi-Piza, K. C.; Sasaki, S. D.; Sampaio, CAM; Tanaka, A. S.; Universidade Federal de São Paulo (UNIFESP); Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)Boophilus microplus trypsin inhibitors (BmTIs) present in larvae were preliminarily characterized as active proteins, approximately 10-18 kDa, by SDS-PAGE. BmTIs showed trypsin inhibitory activity on reverse zymography containing gelatin (0.03%) and also inhibited others serine proteinases (human neutrophil elastase and human plasma kallikrein). Bos indicus, Nelore breed calves, previously sensitized with BmTIs and challenged with tick larvae (20,000 larvae/animal), showed 72.8% efficacy to interfere in tick development with 69.7% and 71.3% reduction of both tick number and egg weight, respectively. Cattle BmTIs antiserum titer was approximately 1:8000. the maximum level of BmTIs antibody production was detected 40 days after the first immunization by ELISA. Our preliminary results suggest that B. microplus serine proteinase inhibitors may play a role in the tick larvae fixation and feeding processes. Therefore, the development of antibodies against BmTIs might impair the normal parasitism. (C) 2002 Elsevier Science B.V. All rights reserved.
- ItemSomente MetadadadosBoophilus microplus tick larvae, a rich source of Kunitz type serine proteinase inhibitors(Elsevier B.V., 2004-09-01) Sasaki, S. D.; Azzolini, SSA; Hirata, I. Y.; Andreotti, R.; Tanaka, A. S.; Universidade Federal de São Paulo (UNIFESP); Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)Serine proteinase inhibitors from Boophilus microplus tick larvae (BmTIs) were purified by affinity chromatography on a trypsin-Sepharose column. BmTIs presented molecular weight between M, 6200 and 18,400 and inhibitory activity for trypsin, HuPK (human plasma kallikrein) and neutrophil elastase. Using ion exchange chromatography, BmTIs were separated in several protein pools named BmTI-A to BmTI-F and BmTI-1 to BmTI-7. All BmTI forms presented inhibitory activity for trypsin with apparent dissociation constants (K-i) in the nM range. in this work, we describe the purification of BmTI-D, BmTI-2, and BmTI-3. These three inhibitors affected neutrophil elastase and HuPK with K-i also in nM range. BMTI-D proved to be the best HuPK inhibitor, while BmTI-3 was more efficient for neutrophil elastase with dissociation constants (K-i) of 12 and 0.5 nM, respectively. BmTI-D. BmTI-2, and BmTI-3 N-terminal amino acid sequences allowed us to include them into the BPTI-Kunitz type serine proteinase inhibitor family. BmTIs purified on trypsin-Sepharose were also used in a bovine immunization assay, resulting in antibody (anti-BmTIs) production. (C) 2004 Elsevier SAS. All rights reserved.
- ItemSomente MetadadadosrBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and biochemical characterization(Elsevier B.V., 2008-08-01) Sasaki, Sergio D.; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade Federal do ABC (UFABC)Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. in this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin. (C) 2008 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosAn unexpected inhibitory activity of Kunitz-type serine proteinase inhibitor derived from Boophilus microplus trypsin inhibitor on cathepsin L(Elsevier B.V., 2006-03-03) Sasaki, S. D.; Cotrin, S. S.; Carmona, A. K.; Tanaka, A. S.; Universidade Federal de São Paulo (UNIFESP)Several BPTI-Kunitz-type serine proteinase inhibitors were described in tick Boophilus microplus and Rhipicephalus sanguineus species. in this work, we present a synthetic gene based on two tick BPTI-Kunitz-type serine proteinase inhibitors, the first domain of B. micro-plus trypsin inhibitor-A (BmTI-A) and the carrapatin, the inhibitors were named BmTIsint and BmTIsint Mut. Our present results showed that BmTIsint and BmTIsint Mut inhibited trypsin (K-i 3.3 and 1.0 nM) and human plasma kallikrein (K-i 16.5 and 35 nM), but in contrast to BmTI-A, the inhibitors did not inhibit human neutrophil elastase. BmTIsint was able to produce immunological response in mice but not in bovines. in addition, it is the first description of a BPTI-Kunitz-type inhibitor as a cysteine proteinase inhibitor, BmTIsint apparent dissociation constant (Ki) for cathepsin L was 108 nM. Our findings open the possibility up to obtain new molecules as potent serine or cysteine proteinase inhibitors using BmTIsint as a model. (c) 2006 Elsevier Inc. All rights reserved.