DIROFILARIA-IMMITIS - MOLECULAR-CLONING AND EXPRESSION OF A CDNA-ENCODING A SELENIUM-INDEPENDENT SECRETED GLUTATHIONE-PEROXIDASE

A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from a Dirofilaria immitis adult female cDNA expression Libra ry by a combination of polymerase chain reaction amplification with pr imers designed from the Brugia pahangi glutathione peroxidase gene seq uence and hybridization screening of D. immitis cDNA libraries. The Di 29 nucleotide and deduced amino acid sequences were very similar to th ose described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for selenoc ysteine in the active site. The cDNA clone was expressed in Escherichi a coli and Spodoptera frugiperda Sf9 insect cells, and the resulting r ecombinant proteins were purified for antibody production and assessme nt of enzymatic properties, respectively. An antiserum generated again st the E. coli-expressed protein detected a protein of 29 kDa in D. im mitis via immunoblotting. This protein is expressed in adult worms (bo th sexes) and fourth stage larvae generated via 6 days of in vitro cul ture, but was undetectable in microfilariae, and third stage larvae ob tained either directly from mosquitoes or following 2 days of culture. The Di29-encoded recombinant protein was secreted from Sf9 insect cel ls and displayed low-level glutathione peroxidase activity against a r ange of hydroperoxide substrates, including hydrogen peroxide. (C) 199 8 Academic Press.