A 2359-base pair DNA fragment from Cryptosporidium parvum encoding a repetitive oocyst protein

Abstract

A Cryptosporidium parvum lambda gt11 expression library was constructed using EcoRI-digested genomic DNA extracted from in vitro-excysted oocysts. Screening of this library with rat anti-Cryptosporidium antiserum led to the isolation of a clone containing a 2359-bp EcoRI fragment. When this fragment was ligated into the EcoRI site of plasmid vector pMS1S, the resulting clone expressed a 200-kDa beta-galactosidase fusion protein. Western blot analysis using serum raised against this fusion protein indicated that the EcoRI fragment represented part of a gene encoding a 190-kDa oocyst wall protein of C. parvum. Sequencing of the fragment revealed a continuous open reading frame encoding 786 amino acids. The DNA sequence is relatively low in G+C (39.1%), and the third codon position contains only 17.9% G+C. The deduced peptide sequence has unusually high proportions of cysteine, proline, glutamine and histidine. Another striking feature of the amino acid sequence is the presence of distinctly repetitive regions based on conserved cysteine residues.