Characterization of cDNA clones encoding a major microneme antigen of Sarcocystis muris (Apicomplexa) cyst merozoites

Abstract

Two monoclonal antibodies directed against a microneme antigen of Sarcocystis muris cyst merozoites (16/17 kDa band doublet) were used to isolate cDNA clones from a lambda ZAP expression library. Restriction analysis revealed that the inserts were highly similar, with sizes ranging between 1.8 and 2.3 kb. In addition, a full-length cDNA insert of 2.6 kb was obtained by hybridization screening. On Northern blots, a single mRNA species of 2.7 kb was detected by a cDNA-derived probe. Southern blot hybridization suggests that the gene is present as a single copy. The nucleotide sequence of the full-length clone contains a single reading frame with a coding capacity of 26.5 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide followed by a hydrophilic domain of unknown function, and the mature protein sequence. After purifying the 16/17 kDa antigen from cyst merozoites, a partial N-terminal amino acid sequence was obtained. Thus, the identity of the cDNA sequence was confirmed. The deduced sequence of the mature protein is predominantly hydrophilic and rich in cysteine (8.7%). Database searching suggested weak homologies of the hypothetical polypeptide to plasma kallikrein, tenascin and blood coagulation factors.