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Antigen encoding gene fragments of Cryptosporidium parvum

<i>Cryptosporidium parvum</i> is an obligate intracellular protozoan which infects the gastrointestinal tract of a wide range of mammalian species. It is a common cause of diarrhoeal illness in humans and neonatal ruminants. Despite the medical and veterinary importance of <i>C. parvum</i> studies of this organism at the genetic level have begun only recently. This is due to the lack of interest shown in the parasite until it was recognised as a cause of human and animal disease, and also to the difficulty in producing sufficient parasite material in order to carry out such studies. The aim of this study was to identify, by screening a DNA library with anti-C. <i>parvum</i> antisera, genes or gene fragments encoding antigens of <i>C.parvum</i>. A <i>C. parvum</i> λgt 11 expression library was constructed using <i>Eco</i>RI-digested genomic DNA prepared from <i>in vitro</i>-excysted oocysts. Screening the library resulted in the isolation of two immunopositive clones, λCPR1, recognised by rat serum raised against excysted <i>C. parvum</i> oocysts, and λCPS10 recognised by serum from a gnotobiotic lamb experimentally infected with <i>C. parvum</i>. The DNA inserts from these clones (CPR1 and CPS10 respectively) were subcloned into the pMS plasmid expression vectors, and the recombinant peptides expressed by the resulting subclones analysed by Western blotting. Subclones containing CPS10 expressed a peptide which was recognised by some, but not all, lambs infected with <i>C. parvum</i>. When CPR1 was subcloned into pMS1S the resulting subclone expressed a 200kDa β-galactosidase fusion protein. This fusion protein was partially purified and used to raise polyclonal antiserum in a rabbit. Western blotting indicated that this serum recognised a 190kDa peptide constituent of the <i>C. parvum</i> oocyst wall. The CPR1 DNA fragment was sequenced in both directions and found to consist of 2359 nucleotides, 2358 of which form a continuous open reading frame. The DNA sequence has a realtively low G+ C content (39.1%) and there is a corresponding bias towards the use of codons ending in A or T (82.1%) within this open reading frame.
Date January 1992
CreatorsLally, Nicola C.
PublisherUniversity of Edinburgh
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation

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