Molecular Characterization of Aminopeptidase N Cloned from Beet Armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Partial cDNA fragments of four homologous genes of aminopeptidase N (APN), known as a putative receptor of Bacillus thuringiensis (Bt) Cry toxin, were identified from Spodoptera exigua midgut using degenerate primers designed from conserved sequence of APN. Among them, one was determined to be predominant, and its full-length cDNA fragment (named as SeAPN) was cloned and characterized. The SeAPN encoding a 1, 007-amino acid protein possessed typical features of APN including signal peptide sequence, potential N-linked glycosylation sites, a zinc-binding metalloprotease consensus motif and GPI-anchor addition sequence. Sequence alignment showed high sequence similarity with other lepidopteran APNs known to be Cry toxin receptor. A full-length SeAPN construct was cloned into pIB/V5-His vector and transfected to Drosophila S2 cells. Enzyme preparation from the S2 cells expressing SeAPN exhibited a substantially high level of aminopeptidase activity, demonstrating its function as APN. Activated Cry toxin cocktail was prepared from B. thuringiensis subsp. aizawai HD-133, which known to be toxic against S. exigua, and used for the cytotoxicity test against the S2 cells expressing SeAPN. However, no symptom of cytotoxicity by the Cry toxin cocktail was observed.