Identification and characterisation of three antigenic proteins from Cryptosporidium parvum sporozoites using a DNA library expressing poly-histidine tagged peptides.

To identify antigenic peptides of the parasite Cryptosporidium parvum, an expression library that allows for the production of chimeric proteins fused with a 6-histidine tag was made. The library was screened with C. parvum sporozoite rabbit anti-serum, and three positive clones (sa20, sa35, and sa40) were identified. The corresponding recombinant proteins (SA20, SA35, and SA40) were expressed in Escherichia coli and purified by metal-affinity chromatography. The sequence of sa20 and sa35 clones did not show any homology with known genes or proteins, whereas the 5' end of the sa40 clone showed homology with two previously identified C. parvum sequences. Hybridisations to intact chromosomes fractionated by pulsed-field gel electrophoresis revealed that the sa35 and sa40 sequences are localised on chromosome VII, whereas the sa20 sequence is localised on chromosome VI. Reverse transcriptase-PCR experiments showed the presence of mRNAs for sa35 and sa40 in the oocyst, whereas the sa20 mRNA was undetectable in this stage. The serological response to the three proteins was assayed in C. parvum-immunised rabbits and in immunocompetent individuals with cryptosporidiosis. The Western blot results indicated that rabbits, challenged with a sporozoite crude antigen or with an oocyst crude antigen, were highly responsive to these three antigens. Human serum samples showed a response to the three proteins, although the response to SA20 appeared to be unrelated to a recent C. parvum infection. These results suggest that the SA35 and the SA40 proteins may be useful in detecting C. parvum infections.