RESUMEN
The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus.
Asunto(s)
Animales , Ratas , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Glutatión Peroxidasa/genética , Enfermedades de las Cabras/parasitología , Cabras , Hemoncosis/parasitología , Haemonchus/enzimología , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Filogenia , ARN de Helminto/química , Técnica del ADN Polimorfo Amplificado Aleatorio , Ratas Sprague-Dawley , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.
Asunto(s)
Animales , Caseínas/metabolismo , Catepsina B , Inhibidores de Cisteína Proteinasa/farmacología , ADN Complementario/genética , Enfermedades de las Cabras/parasitología , Cabras , Hemoncosis/parasitología , Haemonchus/enzimología , Pruebas de Hemaglutinación/veterinaria , Hemoglobinas/metabolismo , Concentración de Iones de Hidrógeno , Inmunoglobulina G/metabolismo , Leucina/análogos & derivados , ARN de Helminto/química , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinariaRESUMEN
A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-senstive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.
Asunto(s)
Animales , Humanos , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Clonorchis sinensis/genética , Proteínas del Helminto/genética , Datos de Secuencia Molecular , Canales de Potasio de Rectificación Interna/genética , ARN de Helminto/química , Alineación de SecuenciaRESUMEN
DNA vaccine against Cysticercus cellulosae infection was developed and its efficacy was tested. A pair of primers specific to antigen B gene of C. cellulosae was designed which amplified the gene successfully with RT-PCR. The gene was ligated to PV93 vector, and the recombinant of antigen B gene and PV93 was transformed to JM83 cells. The transformed JM83 cells were cultured in a large scale and the plasmid purified. Based on the recombinant plasmid. a DNA vaccine was developed and used to vaccinate two groups of experimental pigs. In each group, there was a routine vaccine, an enhanced vaccine and a control group. Groups 1 and 2 were challenged at 4 months and at 14 days post vaccination respectively with eggs of Taenia solium. The antibody response was also tested with ELISA. The results suggested that all animals vaccinated AgB gene DNA vaccine, no matter by routine or enhanced vaccine, their antibodies reached maximum peak 23 days post vaccination and decreased gradually. When the animals were challenged 4 months after vaccination, they had strong immunity and the parasites decrease rates were 91.2% and 93.1% respectively. When pigs vaccinated with AgB gene DNA vaccine were challenged 14 days post vaccination with 18,000 eggs/pig. The animals showed strong immunity and the parasite decrease rates were 99.5% and 84.9% respectively. However at that time, the antibodies did not reach the peak. While in the control group, the number of C. cellulosae was as many as 2,500. It was concluded that the pigs vaccinated with DNA vaccine had strong immunity against infection of eggs of T. solium.