Paramyosin of Echinococcus granulosus: cDNA sequence and characterization of a tegumental antigen.
A lambda ZAPII cDNA library of Echinococcus granulosus larvae was expressed in Escherichia coli SURE cells. Screening of the library with a rabbit antiserum raised against total larval antigen yielded several immunoreactive clones. For analysis of the nucleotide sequence, in vivo excision into pBlueskript was carried out and the 3' end of the cloned insert was sequenced. Three of these clones exhibited identical nucleotide sequences, suggesting expression of identical genes. The complete nucleotide sequence of the largest clone, EG36, with a 3.4-kb insert was determined, presenting an open reading frame of 2.59 kb. The predicted amino acid sequence showed 71.4% identity to the Schistosoma mansoni paramyosin and a significant homology to a 17 amino-acid peptide sequence from antigen B of Taenia solium. From these data we conclude that EG36 is the paramyosin of E. granulosus. For protein purification, the coding sequence of the cDNA was amplified by polymerase chain reaction and ligated in frame into the expression vector pGEX-3X. Affinity-chromatography-purified GST fusion protein was used to induce a polyclonal rabbit antiserum. Immunoblot analysis revealed the expression of a 97-kDa protein by the E. coli clone and that of a protein with a similar molecular weight in protoscolices from E. granulosus and E. multilocularis as well as in E. granulosus cyst fluid. Immunofluorescence studies showed that EG36 was localized throughout the tegument of E. granulosus and E. multilocularis larvae. Sera from patients suffering from echinococcosis, schistosomiasis, and neurocysticercosis reacted with the purified fusion protein when tested in an enzyme-linked immunosorbent assay.
Mühlschlegel
F
F
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Sygulla
L
L
Frosch
P
P
Massetti
P
P
Frosch
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics*; D000954:Antigens, Surface; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D004446:Echinococcus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000235:genetics* / Q000302:isolation & purification
D000947:Antigens, Helminth; D000954:Antigens, Surface; D018076:DNA, Complementary; D014335:Tropomyosin