Cloning and expression of cDNA encoding a cysteine protease inhibitor from clamworm and its possible use in managing Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae)

摘要

A cDNA encoding a cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%, 48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum, and the ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Escherichia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Nicharged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PACPI was 16 kDa as deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. A constitutive and secretive expression vector was also constructed, and the cDNA encoding CPI was subcloned into the vector for extracellular expression. Western blotting analysis results showed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5α harboring pUC18ompAcat- CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5α and control.