Molecular Cloning of an Immunogenic Protein of Baylisascaris procyonis and Expression in Escherichia coli for Use in Developing Improved Serodiagnostic Assays

Abstract

Larva migrans caused by Baylisascaris procyonis is an important zoonotic disease. Current serological diagnostic assays for this disease depend on the use of the parasite's larval excretory-secretory (ES) antigens. In order to identify genes encoding ES antigens and to generate recombinant antigens for use in diagnostic assays, construction and immunoscreening of a B. procyonis third-stage larva cDNA expression library was performed and resulted in identification of a partial-length cDNA clone encoding an ES antigen, designated repeat antigen 1 (RAG1). The full-length rag1 cDNA contained a 753-bp open reading frame that encoded a protein of 250 amino acids with 12 tandem repeats of a 12-amino-acid long sequence. The rag1 genomic DNA revealed a single intron of 837 bp that separated the 753-bp coding sequence into two exons delimited by canonical splice sites. No nucleotide or amino acid sequences present in the GenBank databases had significant similarity with those of RAG1. We have cloned, expressed, and purified the recombinant RAG1 (rRAG1) and analyzed its diagnostic potential by enzyme-linked immunosorbent assay. Anti-Baylisascaris speciesspecific rabbit serum showed strong reactivity to rRAG1, while only minimal to no reactivity was observed with sera against the related ascarids Toxocara canis and Ascaris suum, strongly suggesting the specificity of rRAG1. On the basis of these results, the identified RAG1 appears to be a promising diagnostic antigen for the development of serological assays for specific detection of B. procyonis larva migrans.

Interlaboratory Optimization and Evaluation of a Serological Assay for Diagnosis of Human Baylisascariasis

“…At the NRCP, expression and purification of the rBpRAG1 protein were performed by a procedure modified from the protocol described by Dangoudoubiyam et al. (7). One liter of super optimal broth (SOB) medium with final concentrations of 34 g/ml chloramphenicol and 100 g/ml ampicillin was inoculated with an overnight culture of BL21(DE3)pLysS competent Escherichia coli transformed with the pRSET C/RAG1 expression plasmid.…”

“…The resin-lysate mixture was transferred to a polypropylene column and packed under gravity flow. Six wash buffers with decreasing concentrations of urea (8,7,6,5,4, and 3 M) in 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, 10 mM ␤-mercaptoethanol, and 20 mM imidazole were applied to the column and drained by gravity flow. The bound protein was eluted in 8 column volumes (20 ml) of elution buffer (20 mM Tris-HCl [pH 8.5], 500 mM NaCl, 3 M urea, 10 mM ␤-mercaptoethanol, 150 mM imidazole).…”

“…The pRSET C/RAG1 plasmid was obtained by the CDC and NRCP from Purdue University in 2012. Expression, purification, and Western blot optimization were performed independently at each center, based on the methods described by Dangoudoubiyam et al (7).…”

“…Recently, serological assays for baylisascariasis have improved greatly with the use of a recombinant protein (5,7). An ELISA based on the recombinant Baylisascaris procyonis RAG1 protein (rBpRAG1) was developed, and early testing showed no crossreactivity with Toxocara infections (7).…”

Zoonosis Caused by Baylisascaris procyonis

“…Recently, a recombinant B. procyonis antigen, BpRAG1, was reported for use in development of improved serological assays for the diagnosis of Baylisascaris larva migrans (Dangoudoubiyam et al, 2010). In a recent study, authors tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including 20 patients with clinical Baylisascaris larva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 with unknown/suspected parasitic infections.…”