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KMID : 0545120100200081243
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 8 p.1243 ~ p.1250
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Cloning and Expression of cDNA encoding a Cysteine Protease Inhibitor from clamworm and its possible use in managing Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae)
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Shengnan Li
Daosen Guo
Boguang Zhao
Jianling Ye
Jie Tian
Wenqing Ren
Yunwei Ju
Peng Cui
Ronggui Li
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Abstract
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A cDNA encoding 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 ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Eschechia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 KDa 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.
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KEYWORD
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cysteine protease inhibitor, constitutive and secretive expression vector, Anoplophora glabripennis, cDNA library, Perinereis aibuhitensis Grube
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