Molecular cloning of a serine proteinase inhibitor from Brugia malayi

Abstract

The antigens produced by the infective-stage larvae of filarial parasites are potentially important targets for a protective immune response. A major impediment to studies on the biochemistry and molecular biology of antigens from infective larvae is a lack of parasite material. By employing a reverse transcription PCR-based strategy which exploited the presence of a conserved 22-nucleotide spliced leader sequence present at the 5' end of a proportion of nematode transcripts, spliced leader-containing cDNAs were amplified from the late-vector-stage larvae of the filarial nematode Brugia malayi. A major 1.4-kb PCR product was cloned into pBluescript. One of the PCR cDNA clones (BmY8) contained a 1,287-bp insert that encoded the first member of the serine proteinase inhibitor (serpin) superfamily to be described from nematodes. Reverse transcription PCR analysis of RNA isolated from different developmental stages of the parasite showed that transcription of the B. malayi serpin (Bmserpin) begins between days 8 and 9 of larval development within the insect vector and continues through to the adult and microfilarial stages. In immunoblot analyses of B. malayi somatic extracts, the native protein was estimated to have a molecular weight of 44,000. In immunoblots using excretory-secretory products from infective- and fourth-stage larvae, a single band with an estimated molecular weight of 75,000 was detected. A quantitative analysis of somatic extracts demonstrated that infective-stage larvae contained 10- to 16-fold-more Bmserpin than adults or microfilariae. Bmserpin was immunogenic in gerbils and was recognized strongly by sera from immunized animals. Bmserpin, which has the potential for modifying host defense responses, may play an important role in parasite survival during the early phase of vertebrate-stage development.