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, Papilio xuthus. A full length cDNA of P. xuthus attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete P. xuthus 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 P. xuthus 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 P. xuthus attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature P. xuthus attacin into the expression vector, highly expressed in E. coli BL21 cells, and its antibacterial activity was analyzed. Recombinant P. xuthus attacin evidenced considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35 and Klebsiella pneumonia.

Identification and functional analysis of an iron‐binding protein, ferritin heavy chain subunit, from the swallowtail butterfly, Papilio xuthus

“…The swallowtail butterfly P. xuthus was collected from the orange orchard. All larvae were reared using fresh leaves of Ponkan mandar in clean insect rearing cages under a 16:8 hr (L:D) photocycle at 25℃ and 60–65% relative humidity (Kim et al, ).…”

“…The specific primers are shown in Table . The housekeeping gene P. xuthus ribosomal protein L3 ( RPL3 ) was used as an internal control (Kim et al, ). RT‐qPCR was executed in a Multicolor Real‐time PCR Detection System (Bio‐Rad, Hercules, CA) with a 20 μl reaction mixture containing 10 μl SYBR II, 0.5 μl each of forward primer and reverse primer, 1.0 μl cDNA, and 8 μl Rnase‐free H 2 O.…”