Cloning and expression of cDNA encoding a cysteine protease inhibitor from clamworm and its possible use in managing Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae).
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 Escherichia 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 DH5alpha harboring pUC18ompAcat-CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5alpha and control.
Li
Shengnan
S
Department of Biology, Qingdao University, Qingdao, P.R. China.
Guo
Daosen
D
Zhao
Boguang
B
Ye
Jianling
J
Tian
Jie
J
Ren
Wenqing
W
Ju
Yunwei
Y
Cui
Peng
P
Li
Ronggui
R
D000595:Amino Acid Sequence; D000818:Animals; D000845:Annelida / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007300:Insect Control; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment
D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D007306:Insecticides; D011994:Recombinant Proteins