Isolation, identification and expression of a Fasciola hepatica cDNA encoding a 2.9-kDa recombinant protein for the diagnosis of ovine fasciolosis.
A 400-bp Fasciola hepatica cDNA clone was isolated from an expression library by immunological screening using rat sera taken 2 weeks after experimental infection. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 78 bp which encoded a 25 amino acid polypeptide with a predicted molecular weight of 2.9 kDa. This polypeptide was expressed in bacteria as a GST-fusion protein and used for the production of specific antigen. The 2.9 kDa recombinant protein (APS) was evaluated against sera from experimentally infected sheep using an indirect ELISA, and the results were compared to those obtained using F. hepatica excretory/secretory products (ESP). The pattern of IgG was very similar both against the recombinant and the native proteins, increasing early following the infection. After treatment with triclabendazole, the IgG response against the APS seroreverted to negative values, whereas it remained elevated against the ESP. We conclude that this recombinant protein could be used in diagnostic assays for the identification of recently infected sheep.
Paz-Silva
A
A
Departamento de Patología Animal, Parasitología y Enfermedades Parasitarias y Epidemiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27071 , Lugo, Spain. adolpaz@lugo.usc.es
Hillyer
G V
GV
Sánchez-Andrade
R
R
Rodríguez-Medina
J R
JR
Arias
M
M
Morrondo
P
P
Díez-Baños
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018509:DNA, Helminth / Q000235:genetics* / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005210:Fasciola hepatica / Q000235:genetics*; D005211:Fascioliasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D015870:Gene Expression; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000469:parasitology
D000909:Antibodies, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D011994:Recombinant Proteins