cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica.

A gamma gt cDNA library was constructed from poly(U)-Sepharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens

B. E. Torian; B. M. Flores; V. L. Stroeher; F. S. Hagen; W. E. Stamm

2006

Scholarcy highlights

  • A gamma gt cDNA library was constructed from poly(U)-Sepharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens
  • The library was screened with rabbit polyclonal anti-E. histolytica serum
  • DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis
  • A 29-kDa protein was identified as a surface antigen when monoclonal antibodies were used to immunoprecipitate the antigen from metabolically 35S-labeled live trophozoites
  • The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-125I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degrees C
  • Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

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