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

This research article focuses on the identification, sequencing, and expression of a gene encoding a key surface antigen of Sarcocystis neurona, a parasite mainly responsible for a neurological disease known as equine protozoal myeloencephalitis (EPM) in horses. It highlights the cloning of this gene, its expression as a recombinant protein, and its significant recognition by relevant antibodies, calling this antigen SnSAG1.

Construction of the cDNA Library and Identification of the Antigen-encoding Gene

• The researchers started by preparing a complementary DNA (cDNA) library. This cDNA library was constructed from polyA+mRNA isolated from S. neurona merozoites grown in vitro, using the lambda ZAP expression vector.
• They randomly sequenced 96 clones and identified a clone that was abundant. This clone’s translated amino acid sequence showed a 30% identity with the 31-kDa surface antigen of Sarcocystis muris cyst merozoites. This similarity indicated that the identified clone was likely encoding the major 29-kDa surface antigen of S. neurona.

Analyses of the Antigen-Encoding Gene

• Further research via Southern blot analysis showed that the corresponding gene exists in low copy number within the S. neurona genome. However, RNA blot analysis and additional data suggested that the gene transcript is highly abundant.
• The sequence of the identified 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 included a potential N-terminal signal peptide sequence followed by a polypeptide with 12 cysteines.

Expression of the Recombinant Protein and Antigen Recognition

• The coding region of the cDNA insert was subcloned into the expression vector pET14b, leading to the expression of a fusion protein.
• This recombinant polypeptide was recognised by two monoclonal antibodies, mAb 2A7 and mAb 1631. These antibodies target a native 29-kDa protein found on the surface of cultured S. neurona merozoites.
• Furthermore, antibodies in serum and cerebrospinal fluid from a horse with EPM also recognised the native 29-kDa protein of S. neurona merozoites and the 29-kDa recombinant protein.

Naming of the Surface Antigen

Based on these findings, the researchers named this S. neurona surface antigen SnSAG1. This study thus provides a valuable reference for future research on EPM, particularly for the development of diagnostic and therapeutic approaches.