RESUMO
Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD600nm = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment.
Assuntos
Escherichia coli , Expressão Gênica , Aranhas/genética , Animais , Antivenenos/biossíntese , Antivenenos/genética , Antivenenos/imunologia , Antivenenos/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos Endogâmicos BALB C , Diester Fosfórico Hidrolases/biossíntese , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Diester Fosfórico Hidrolases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Venenos de Aranha/biossíntese , Venenos de Aranha/genética , Venenos de Aranha/imunologia , Venenos de Aranha/isolamento & purificaçãoRESUMO
Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded ß-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells.
Assuntos
Proteínas de Artrópodes/biossíntese , Nucleopoliedrovírus , Venenos de Aranha/biossíntese , Aranhas/genética , Animais , Proteínas de Artrópodes/genética , Linhagem Celular , Necrose/genética , Necrose/metabolismo , Necrose/patologia , Venenos de Aranha/genética , SpodopteraRESUMO
Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.
Assuntos
Biomarcadores Tumorais/genética , Venenos de Aranha/genética , Aranhas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/química , Biomarcadores Tumorais/imunologia , Permeabilidade Capilar/efeitos dos fármacos , Clonagem Molecular , Reações Cruzadas , Edema/etiologia , Camundongos , Dados de Sequência Molecular , Coelhos , Venenos de Aranha/biossíntese , Venenos de Aranha/química , Venenos de Aranha/imunologia , Aranhas/genética , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels.
Assuntos
Neurotoxinas/biossíntese , Neurotoxinas/farmacologia , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Animais , Cálcio/fisiologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Clonagem Molecular , Receptores de Inositol 1,4,5-Trifosfato/biossíntese , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Canal de Liberação de Cálcio do Receptor de Rianodina/biossíntese , Venenos de Aranha/biossíntese , Aranhas/químicaRESUMO
A cDNA library was constructed from the venom glands of the Brazilian "armed" spider, Phoneutria nigriventer, and a clone coding for Tx1, a lethal toxin, was identified and sequenced. The sequence data derived from this cDNA clone combined with the previously determined amino acid sequence predict that Tx1 is initially synthesized as a preprotoxin. Four segments (comprising the signal sequence, a short, 15-amino acid, glutamate-rich sequence, the functional toxin, and 2 glycine residues) can be distinguished. The structure of the preprotoxin and the proposed processing steps required to form the mature Tx1 toxin show similarities with the synthesis and processing of omega-agatoxin IA.