Abstract: 【Aim】 The aim of this study is to analyze the cloning, expression and homology modeling of midgut aminopeptidase gene PxAPN5 in the diamondback moth, Plutella xylostella. 【Methods】 A aminopeptidase (APN) gene was cloned from the P. xylostella midgut, and bioinformatics analysis of the gene was performed. The APN protein was expressed using prokaryotic expression system, and its enzymatic activity was assayed. The interaction between APN and Cry2Ab was determined by using ligand blot analysis. Homology modeling of APN gene was conducted for the prediction of characteristics and functions of mutation sites. 【Results】 The sequencing results showed that the cloned APN gene (NCBI accession no.: KM034756) is 2 853 bp in length and encodes 950 amino acids with the predicted molecular weight of 107.3871 kDa and isoelectric point of 5.24. Phylogenetic tree analysis indicated that this APN gene belongs to class 5 of APN family, and it was named as PxAPN5. PxAPN5 has the conservative features of the APN proteins in lepidopteran insects including N-glycosylation and O-glycosylation sites, GPI anchor point, C-transmembrane domains and zinc-metalloprotease domain (³⁶¹HEXXH³⁶⁵). A 110 kDa specific protein band appeared when APN protein was inducibly expressed in Escherichia coli. Aminopeptidase activity assay showed that the supernatant of broken bacteria possessed aminopeptidase activity and its specific activity was 1 047.2 U/g. The ligand blot result indicated that PxAPN5 could bind to Cry2Ab specially. Multiple alignment of amino acid sequences demonstrated that there are three mutations in PxAPN5 when compared to other known APN proteins from P. xylostella. 【Conclusion】 The PxAPN5 protein with aminopeptidase activity was successfully cloned and expressed, and it could bind to Cry2Ab. Prediction of characteristics and functions of mutation sites in PxAPN5 was carried out by homology modeling. These results laid the foundation for the functional research of PxAPN.

Key words:  Plutella xylostella, aminopeptidase (APN) gene, gene analysis, prokaryotic expression, enzyme activity, ligand blot, homology modeling