Molecular Cloning and Characterization of Attacin from the Swallowtail Butterfly, Papilio xuthus

Abstract

Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, <TEX>$Papilio$</TEX> <TEX>$xuthus$</TEX>. A full length cDNA of <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin into the expression vector, highly expressed in <TEX>$E.$</TEX> <TEX>$coli$</TEX> BL21 cells, and its antibacterial activity was analyzed. Recombinant <TEX>$P.$</TEX> <TEX>$xuthus$</TEX> attacin evidenced considerably antibacterial activity against Gram-negative bacteria, <TEX>$E.$</TEX> <TEX>$coli$</TEX> ML 35 and <TEX>$Klebsiella$</TEX> <TEX>$pneumonia$</TEX>.