Identification and molecular characterisation of a putative gene for cell detaching factor from Trichomonas vaginalis.
|Title:||Identification and molecular characterisation of a putative gene for cell detaching factor from Trichomonas vaginalis.|
|Authors:||Delgaty, Kiera L.|
|Abstract:||Trichomonas vaginalis (Tv) is the etiologic agent of trichomoniasis, a sexually transmitted disease of significant socio-economic importance. Cell-free filtrate from Tv growth media causes rounding and detachment of adherent monolayers of human epithelial cells in culture. Cell detaching factor (CDF), a 200 kDa glycoprotein in the purified preparation of the cell-free filtrate, is believed to play an important role in the pathogenesis of Tv. The identification of the gene for CDF would therefore allow further characterisation of the protein and its expression. A cDNA clone (CDF-2) was identified in a Tv cDNA library by immunological screen with rabbit anti-serum prepared against a purified preparation of CDF. This clone contained an open reading frame (ORF) that was believed to represent a partial coding sequence for CDF. A genome walk initiated from CDF-2 using Rapid Amplification of Genomic DNA Ends (RAGE) identified a contiguous 6.5 kb ORF Northern analysis, which identified a 6.5--7 kb transcript, supported the size of the ORF, while Rapid Amplification of cDNA Ends (RACE) identified a putative 5' end of the ORF and identified a 31 bp 3' untranslated region. The ORF is predicted to encode a 232 kDa polypeptide containing 42 EEKPKL repeats, but has no sequence homology with any previously reported protein. The full-length ORF was amplified from Tv genomic DNA by PCR and cloned into the plasmid vector pCR2.1-TOPO. The ORF was transcribed in vitro using T7 RNA polymerase and translated in rabbit reticulocyte lysate in the presence of 35S-methionine. Several high molecular weight proteins (>200, 165, 150, 130, 118, 80, 47 kDa) were produced. Two polypeptides of 205 and 220 kDa were immunoprecipitated from the in vitro translation mix by the anti-CDF serum. These data suggest that the gene encoding Tv CDF has been identified.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|