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( Sheng Nan Li ),( Dao Sen Guo ),( Bo Guang Zhao ),( Jian Ling Ye ),( Jie Tian ),( Wen Qing Ren ),( Yun Wei Ju ),( Peng Cui ),( Rong Gui Li ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.8
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.