Molecular cloning and phylogenetic analysis of Clonorchis sinensis elongation factor-1alpha.
Elongation factor-1 (EF-1) plays a primary role in protein synthesis, e.g., in the regulation of cell growth, aging, motility, embryogenesis, and signal transduction. The authors identified a clone CsIH23 by immunoscreening a Clonorchis sinensis cDNA library. The cDNA of CsIH23 was found to have a putative open reading frame containing 461 amino acids with a predicted molecular mass of 50.5 kDa. Its polypeptide sequence was highly homologous with EF-1alpha of parasites and vertebrate animals. CsIH23 polypeptide contained three GTP/GDP-binding sites, one ribosome-binding domain, one actin-binding domain, one tRNA-binding domain, and two glyceryl-phosphoryl-ethanolamine attachment sites. Based on these primary and secondary structural similarities, it was concluded that CsIH23 cDNA encodes C. sinensis EF-1alpha (CsEF-1alpha). In a molecular phylogenic tree, CsEF-1alpha clustered with the EF-1alpha of helminthic parasites. Subsequently, CsEF-1alpha recombinant protein was bacterially overexpressed and purified by Ni-NTA affinity column chromatography. Immunoblotting using CsEF-1alpha recombinant protein produced positive signals for all serum samples tested from clonorchiasis, opisthorchiasis viverinii, and paragonimiasis westermani patients and normal healthy controls. These findings suggest that recombinant CsEF-1alpha is of limited usefulness as serodiagnostic antigen for clonorchiasis.
Department of Infection Biology, College of Medicine, Chungnam National University, Daejeon 301-131, South Korea.
Tae Yun
TY
Kim
Pyo Yun
PY
Cho
Jong Won
JW
Na
Sung-Jong
SJ
Hong
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood*; D003001:Cloning, Molecular; D003003:Clonorchiasis / Q000175:diagnosis* / Q000469:parasitology; D003004:Clonorchis sinensis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000909:Antibodies, Helminth; D018076:DNA, Complementary; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins