Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein.
A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.
Ruiz de Eguino
A D
AD
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias (CSIC), Universidad de Oviedo, Spain.
Machín
A
A
Casais
R
R
Castro
A M
AM
Boga
J A
JA
Martín-Alonso
J M
JM
Parra
F
F
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000737:chemistry; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005210:Fasciola hepatica / Q000737:chemistry / Q000235:genetics*; D017238:Genes, Helminth; D015801:Helminth Proteins; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011817:Rabbits; D017422:Sequence Analysis, DNA
D000947:Antigens, Helminth; D002135:Calcium-Binding Proteins; C121452:Fh22 protein, Fasciola hepatica; D015801:Helminth Proteins; D002118:Calcium