RESUMO
beta-N-Acetylhexosaminidase (HEX, E.C. 3.2.1.52) from larvae of the ixodid tick Boophilus microplus was purified to capillary zone electrophoresis homogeneity, and characterized. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-200, p-aminobenzyl-N-acetyl-beta-D-thioglucosamine affinity, and Mono-Q FPLC columns. Purification was about 1600-fold, with a yield of 10%, as determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme presented optimum pH 4.7, and optimum temperature 65 degrees C. The molecular weight of non-denatured enzyme was estimated as 127,000 by gel filtration chromatography, and 60,000 in SDS-PAGE. The tick hexosaminidase presented glycosyl residues, as evidenced by binding to Concanavalin-A. Among several p-nitrophenyl glycosides tested as substrate, HEX was active only on p-nitrophenyl-N-acetylglucosaminide and p-nitrophenyl-N-acetylgalactosaminide. The purified enzyme presented immunogenicity in rabbit, and the correspondent antibodies inhibited about 90% of its original, in vitro activity.
Assuntos
Carrapatos/enzimologia , beta-N-Acetil-Hexosaminidases/química , Animais , Anticorpos/farmacologia , Bovinos , Cromatografia , Eletroforese Capilar , Estabilidade Enzimática , Glicosídeos/metabolismo , Cinética , Larva/enzimologia , Metais/farmacologia , Especificidade por Substrato , Carrapatos/embriologia , Carrapatos/parasitologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/isolamento & purificaçãoRESUMO
A polyclonal antibody (anti-HEX) was developed against a soluble N-acetylhexosaminidase (HEX) isolated from larval extracts of Boophilus microplus. Purified hexosaminidase was strongly inhibited by the IgG fraction of this antibody. The antibody inhibited the hexosaminidase activity of other sources, such as haemolymph and larval membranes. The antibody reacted with different antigens in the tick haemolymph, but did not recognize any antigen in saliva, as seen by immunoblot analysis. The anti-HEX was inoculated into fully engorged B. microplus females, resulting in a decrease in oviposition of approximately 26%, relative to the effect of pre-immune IgG. These data show the potential of the use of this tick enzyme as an antigen in vaccine development.
Assuntos
Anticorpos/imunologia , Doenças dos Bovinos/prevenção & controle , Infestações por Carrapato/veterinária , Carrapatos/fisiologia , beta-N-Acetil-Hexosaminidases/imunologia , Animais , Western Blotting/veterinária , Bovinos , Cromatografia em Agarose/veterinária , Cromatografia em Gel/veterinária , Compostos Cromogênicos/química , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Hemolinfa/enzimologia , Imunoglobulina G/imunologia , Oviposição/imunologia , Coelhos , Infestações por Carrapato/prevenção & controle , Carrapatos/imunologia , beta-N-Acetil-Hexosaminidases/análiseRESUMO
The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. The knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle.
Assuntos
Anticorpos/sangue , Doenças dos Bovinos , Hemolinfa/imunologia , Imunoglobulinas/sangue , Infestações por Carrapato/veterinária , Carrapatos/imunologia , Vacinação , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática , Infestações por Carrapato/imunologia , Infestações por Carrapato/prevenção & controleRESUMO
The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.
Assuntos
Proteínas do Ovo/metabolismo , Carrapatos/metabolismo , Animais , Bovinos , Cromatografia por Troca Iônica , Sistema Digestório , Feminino , Larva , Óvulo/metabolismo , Peptídeos/metabolismo , Carrapatos/crescimento & desenvolvimentoRESUMO
Babesia bovis is an intraerythrocytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from two isolates of B. bovis reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites were fractionated in a CsCl buoyant density gradient. A sample containing the 5.5-kb RNA was analysed under an electron microscope and a virus-like particle was observed.