Dirofilaria immitis: molecular cloning and expression of a cDNA encoding a selenium-independent secreted glutathione peroxidase

Abstract

A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from a Dirofilaria immitis adult female cDNA expression library by a combination of polymerase chain reaction amplification with primers designed from the Brugia pahangi glutathione peroxidase gene sequence and hybridization screening of D. immitis cDNA libraries. The Di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for selenocysteine in the active site. The cDNA clone was expressed in Escherichia coli and Spodoptera frugiperda Sf9 insect cells, and the resulting recombinant proteins were purified for antibody production and assessment of enzymatic properties, respectively. An antiserum generated against the E. coli-expressed protein detected a protein of 29 kDa in D. immitis via immunoblotting. This protein is expressed in adult worms (both sexes) and fourth stage larvae generated via 6 days of in vitro culture, but was undetectable in microfilariae, and third stage larvae obtained either directly from mosquitoes or following 2 days of culture. The Di29-encoded recombinant protein was secreted from Sf9 insect cells and displayed low-level glutathione peroxidase activity against a range of hydroperoxide substrates, including hydrogen peroxide.