Molecular characterisation of a major 29 kDa surface antigen of Sarcocystis neurona

Abstract

A gene encoding a major 29 kDa surface antigen from Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), was cloned, sequenced, and expressed as a recombinant protein. A cDNA library was prepared in the expression vector lambda ZAP from polyA+mRNA isolated from S. neurona merozoites cultivated in vitro. Random sequencing of 96 clones identified a clone of an abundant transcript having a translated amino acid sequence with 30% identity to the 31-kDa surface antigen of Sarcocystis muris cyst merozoites. Southern blot analysis indicated that the corresponding gene exists in low copy number within the S. neurona genome, but RNA blot analysis and other data indicated that the gene transcript is highly abundant. The sequence of the cDNA clone encoded an open reading frame specifying a polypeptide of 276 amino acids with a predicted size of 28.7 kDa. The deduced amino acid sequence displayed a hypothetical N-terminal signal peptide sequence followed by a polypeptide containing 12 cysteines. The coding region of the cDNA insert was subcloned into the expression vector pET14b, and a fusion protein expressed. The recombinant polypeptide was recognised by mAb 2A7 and mAb 1631, directed against a 29 kDa native protein found on the surface of cultured merozoites. Antibodies in serum and cerebrospinal fluid from a horse with EPM recognised a 29 kDa native protein of S. neurona merozoites and the 29 kDa recombinant protein. This S. neurona surface antigen is named SnSAG1.