Attacin is an antibacterial protein that is secreted by fat body cells of insect larva in response to bacterial infection. A 949 bp cDNA encoding the antibacterial protein attacin was isolated by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from immunized Papilio xuthus larvae. The attacin cDNAs encoded 250 amino acid residues open reading frame with 60 residues prepropeptide. The deduced amino acid sequence of P. xuthus attacin showed significant identities with other Lepidopteran attacins. The attacin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The predicted mature attacin was expressed as soluble fusion protein efficiently in bacterial expression system. To increase productivity and solubility, attacin was translationally fused with thioredoxin (Trx) protein and expressed in E. coli cells that are highly sensitive to the mature attacin. The recombinant attacin exhibited antibacterial activity against Gram-negative bacteria.

We have previously shown that the larvae of swallowtail butterfly, Papilio xuthus, exhibit substantial antibacterial activity in the hemolymph, upon challenging with bacterial lipopolysaccharide (LPS). Here we report the isolation and molecular characterization of several immune inducible genes that are specifically expressed by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from P. xuthus the larva. Using 120 arbitrary ACPs, we identified 24 differentially expressed genes (DEGs) that are up-regulated in response to injected LPS. Sequence analysis showed that 18 DEGs revelaed a high sequence similarity to the previously characterized genes of other insects, although 6 DEGs showed no significant similarity to any known genes. Among these inducible transcripts we found 8 putative immune-related genes including cecropin and attacin. Finally, we analysed the expression profiles of potential immune-related genes by RT-PCR and found all of them were considerably increased in the mRNA levels by LPS injection.