RESUMEN
Tx1 from the venom of the Brazilian spider, Phoneutria nigriventer, is a lethal neurotoxic polypeptide of M(r) 8600 Da with 14 cysteine residues. It is a novel sodium channel blocker which reversibly inhibits sodium currents in CHO cells expressing recombinant sodium (Nav1.2) channels. We cloned and expressed the Tx1 toxin as a thioredoxin fusion product in the cytoplasm of Escherichia coli. After semipurification by immobilized Ni-ion affinity chromatography, the recombinant Tx1 was purified by reverse phase chromatography and characterized. It displayed similar biochemical and pharmacological properties to the native toxin, and it should be useful for further investigation of structure-function relationship of Na channels.
Asunto(s)
Neuropéptidos/aislamiento & purificación , Bloqueadores de los Canales de Sodio/aislamiento & purificación , Canales de Sodio/efectos de los fármacos , Venenos de Araña/genética , Arañas/genética , Secuencia de Aminoácidos , Animales , Unión Competitiva/efectos de los fármacos , Brasil , Células CHO , Clonación Molecular , Cricetinae , Inyecciones Intraventriculares , Ratones , Datos de Secuencia Molecular , Neuropéptidos/biosíntesis , Neuropéptidos/toxicidad , Unión Proteica , Ratas , Ratas Wistar , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/toxicidad , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/metabolismo , Bloqueadores de los Canales de Sodio/toxicidad , Canales de Sodio/metabolismo , Especificidad de la Especie , Venenos de Araña/química , Relación Estructura-ActividadRESUMEN
Ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of Anopheles aconitus mosquitoes were examined to investigate intra- and inter-species variation amongst the members of the Minimus group of Anopheles subgenus Cellia. Three rDNA loci (ITS1, ITS2 and D3 regions) and a mtDNA locus (cytochrome oxidase II) were analyzed in An. aconitus Form B and Form C collected in Chiang Mai Province, Thailand. The results show that the consensus sequences of the four loci of the two forms are consistent with those of mosquitoes in the genus Anopheles. No intraindividual variation was detected, but intrapopulation variation was present with polymorphic sequences in some forms for each gene examined. The variation rates were approximately 0.15 to 0.8%. These data indicate that An. aconitus Form B and Form C in Chiang Mai, Thailand are conspecific. In this study, the complete ITS1 sequence of An. aconitus is reported for the first time. The region showed a high variation rate (approximately 55%), compared to the closely related species An. minimus C. It is suggested that this rDNA locus may provide sequence information to differentiate the members of the Minimus group of Anopheles subgenus Cellia.