A highly divergent 33 kDa Cryptosporidium parvum antigen

Previous studies comparing the genome sequences of Cryptosporidium parvum with Cryptosporidium hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, the C. parvum DNA sequence cgd8-5370, which encodes a protein whose amino acid sequence differs appreciably from its homologue in C. hominis , was cloned by PCR and expressed as a recombinant protein in Escherichia coli . Antisera raised against the recombinant cgd8-5370 antigen strongly recognized a unique 33 kDa protein in immunoblots from reducing and non-reducing SDS-PAGE of native C. parvum protein. However, anti-Cp33 sera did not recognize the native 33 kDa homologue in C. hominis . In an immunofluorescence assay (IFA), anti-Cp33 serum recognized an antigen in the anterior end of air-dried C. parvum sporozoites but failed to bind at any sites in C. hominis sporozoites, indicating its specificity for C. parvum . IFA staining of live C. parvum sporozoites with anti-Cp33 serum failed to bind to the parasite, indicating that the CP33 antigen is not on the sporozoite surface, which is consistent with topology predictions based on the encoded amino acid sequence. RT-PCR analysis of cgd8-5370 mRNA before or during C. parvum oocyst excystation revealed transcripts only in excysting sporozoites. Thus, Cp33 represents one of a small number of proteins shown to differentiate C. parvum from C. hominis sporozoites and oocysts.