MOLECULAR-CLONING AND CHARACTERIZATION OF A DEVELOPMENTALLY-REGULATEDPUTATIVE METALLOPEPTIDASE PRESENT IN A HOST PROTECTIVE EXTRACT OF HAEMONCHUS-CONTORTUS

Antisera from lambs immunised with the Haemonchus contortus integral m embrane protein complex, Haemonchus galactose-containing glycoprotein (H-gal-GP), the lambs being refractory to subsequent challenge, were u sed to identify several clones from an adult H. contortus lambda gt11 cDNA library. Using gene-specific oligonucleotide primers in conjuncti on with primers directed to a conserved nematode Spliced Leader (SL) s equence and to the polyA(+) tail of mRNA, the remaining 5' and 3' sequ ences of one of these clones, metallopeptidase-l (MEP1), were amplifie d. The 2.4 kb full-length coding sequence was subsequently amplified i n a single reaction. Sequence analysis identified MEP1 as encoding a p utative zinc metallopeptidase, which shared limited homology with the mammalian type II integral membrane protein neutral endopeptidase (NEP ). Southern blotting indicated that MEP1 belonged to a multigene famil y. MEP1 was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, and a specific antiserum raised in sheep. This antise rum recognised several polypeptide components of H-gal-GP. Immunolocal isation studies showed that MEP1 encoded a protein located on the lumi nal surface of the nematode gut. Both MEP1 mRNA and protein are develo pmentally regulated with expression being limited to the blood-feeding stages of H. contortus. (C) 1997 Elsevier Science B.V.