Molecular Cloning and Characterization of a Gene Encoding a 13.1 kDa Antigenic Protein of Naegleria fowleri

Abstract

An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies

Immunological Characterizations of a Cloned 13.1-Kilodalton Protein from Pathogenic Naegleria fowleri

“…Using the immunoscreening technique with infected and immune mouse sera as a first step toward the production of antigenic molecules, Shin et al previously cloned an antigenrelated gene from a cDNA expression library of N. fowleri and named it the nfa1 gene (17). The nfa1 gene consisted of 357 bases that translated into 119 amino acid residues which produced a 13.1-kDa recombinant protein.…”

“…The nfa1 gene consisted of 357 bases that translated into 119 amino acid residues which produced a 13.1-kDa recombinant protein. In Western blotting experiments, the recombinant protein (rNfa1) reacted strongly with infected and immune sera and the anti-Nfa1 antibody (17). As a second step, to get more information on the rNfa1 protein we carried out some immunological studies with an anti-Nfa1 polyclonal antibody.…”

“…To obtain a recombinant Nfa1 fusion protein, expression and purification of the gene product were performed according to a method described in a previous paper (17). The purified DNA (5 g/l) obtained using PCR-T7/NT TOPO expression vector (Invitrogen, Groningen, The Netherlands) containing the nfa1 gene was subsequently used to transform the Escherichia coli strain BL21(DE3)pLysS by the heat shock method.…”

“…Immune and infected sera were prepared according to a method described in a previous paper (17). In addition, sectioned brain tissue for use in immunocytochemistry experiments was obtained from a mouse prepared for infected-serum collection.…”

“…An enzyme-linked immunosorbent assay (ELISA) was performed with purified Nfa1 protein (5 g/ml) and rabbit anti-mouse whole immunoglobulin (1: 10,000 dilution) conjugated with alkaline phosphate (Sigma). Western blotting against recombinant Nfa1 protein was performed according to a method described in a previous paper (17).…”